Antibodies for epidermal growth factor receptor 3 (her3)

ABSTRACT

This invention relates to antibodies or fragments thereof which interact with HER family of receptors, e.g., HER3 receptor. In particular, it relates to antibodies or fragments thereof that recognize a conformational epitope of HER3 receptor comprising residues from both domains 2 and 4 resulting in inhibition of both ligand-dependent and ligand-independent signal transduction; and allow ligand binding (e.g., neuregulin), whilst preventing ligand-induced activation of signal transduction. These antibodies or fragments can be used to treat a number of diseases or disorders characterized by increased levels of HER3 expression (e.g., esophageal cancer). These antibodies or fragments can be used to treat a number of diseases or disorders characterized by the antibodies or fragments ability to decrease tissue weight (e.g., prostate or uterine weights) or to induce atrophy of tissue (e.g., atrophy of male breast).

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/115,330, filed on Aug. 28, 2018, which is a continuation of U.S.patent application Ser. No. 14/852,003, filed on Sep. 11, 2015 and nowU.S. Pat. No. 10,080,800, which claims priority to U.S. patentapplication Ser. No. 13/693,330, filed on Dec. 4, 2012 and now U.S. Pat.No. 9,192,663 which claims priority to U.S. Provisional Application No.61/566,890 filed on Dec. 5, 2011, the contents of which are incorporatedherein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 28, 2018, isnamed PAT054912-US-CNT02_SL and is 380,057 bytes in size.

FIELD OF THE INVENTION

This invention relates generally to antibodies or fragments thereofwhich interact with HER family of receptors, e.g., HER3 receptor. Inparticular, it relates to antibodies or fragments thereof that recognizea conformational epitope of HER3 receptor comprising residues from bothdomains 2 and 4 resulting in inhibition of both ligand-dependent andligand-independent signal transduction; and allow ligand binding (e.g.,neuregulin), whilst preventing ligand-induced activation of signaltransduction. These antibodies or fragments can be used to treat anumber of diseases or disorders characterized by increased levels ofHER3 expression (e.g., esophageal cancer). These antibodies or fragmentscan be used to treat a number of diseases or disorders characterized bythe antibodies or fragments ability to decrease tissue weight (e.g.,prostate or uterine weights) or to induce atrophy of tissue (e.g.,atrophy of male breast).

BACKGROUND OF THE INVENTION

The human epidermal growth factor receptor 3 (ErbB3, also known as HER3)is a receptor protein tyrosine kinase and belongs to the epidermalgrowth factor receptor (EGFR) subfamily of receptor protein tyrosinekinases, which also includes EGFR (HER1, ErbB1), HER2 (ErbB2, Neu), andHER4 (ErbB4) (Plowman et al., (1990) Proc. Natl. Acad. Sci. U.S.A.87:4905-4909; Kraus et al., (1989) Proc. Natl. Acad. Sci. U.S.A.86:9193-9197; and Kraus et al., (1993) Proc. Natl. Acad. Sci. U.S.A.90:2900-2904). Like the prototypical epidermal growth factor receptor,the transmembrane receptor HER3 consists of an extracellularligand-binding domain (ECD), a dimerization domain within the ECD, atransmembrane domain, an intracellular protein tyrosine kinase-likedomain (TKD) and a C-terminal phosphorylation domain. Unlike the otherHER family members, the kinase domain of HER3 displays very lowintrinsic kinase activity.

The ligands neuregulin 1 (NRG) or neuregulin 2 bind to the extracellulardomain of HER3 and activate receptor-mediated signaling pathway bypromoting dimerization with other dimerization partners such as HER2.Heterodimerization results in activation and transphosphorylation ofHER3's intracellular domain and is a means not only for signaldiversification but also signal amplification. In addition, HER3heterodimerization can also occur in the absence of activating ligandsand this is commonly termed ligand-independent HER3 activation. Forexample, when HER2 is expressed at high levels as a result of geneamplification (e.g. in breast, lung, ovarian or gastric cancer)spontaneous HER2/HER3 dimers can be formed. In this situation theHER2/HER3 is considered the most active ErbB signaling dimer and istherefore highly transforming.

Increased HER3 has been found in several types of cancer such as breast,lung, gastrointestinal and pancreatic cancers. Interestingly, acorrelation between the expression of HER2/HER3 and the progression froma non-invasive to an invasive stage has been shown (Alimandi et al.,(1995) Oncogene 10:1813-1821; DeFazio et al., (2000) Cancer 87:487-498;Naidu et al., (1988) Br. J. Cancer 78:1385-1390). Accordingly, agentsthat interfere with HER3 mediated signaling are needed.

SUMMARY OF THE INVENTION

The invention is based on the discovery of antigen binding proteins(e.g., antibodies or fragments thereof) that bind to a conformationalepitope of HER3 receptor comprising amino acid residues within domain 2and domain 4 of HER3. This binding of the antibodies or fragmentsthereof with domain 2 and domain 4 stabilizes the HER3 receptor in aninactive or closed conformation such that HER3 activation is inhibited.Surprisingly, binding of the antibodies or fragments thereof with thisconformational epitope blocks both ligand-dependent (e.g. neuregulin)and ligand-independent HER3 signaling pathways. Furthermore, antibodymediated inhibition of ligand induced signaling occurs without blockingligand binding (i.e. both ligand and antibody can bind HER3) presumablybecause HER3 cannot undergo the conformational rearrangements requiredfor activation. Also disclosed are methods for using the antibodies orfragments thereof to treat a number of diseases or disorderscharacterized by increased expression of HER3; and disease or disorderscharacterized by the antibodies or fragments ability to decrease tissueweight (e.g., prostate or uterine weights) or to induce atrophy oftissue (e.g., atrophy of male breast).

Accordingly, in one aspect, the invention pertains to a method oftreating a disorder characterized by increased levels of HER3 expressionin an esophageal tract comprising: selecting patient suffering fromincreased levels of HER3 expression in an esophageal tract; andadministering an antibody or fragment thereof that specifically binds toa HER3 receptor, such that the antibody or fragment thereof binds to aconformational epitope comprising amino acid residues within domain 2and domain 4 of the HER3 receptor and blocks both ligand-dependent andligand-independent signal transduction, thereby treating the disorder.In one embodiment, the disorder is selected from the group consisting ofesophageal cancer and Barretts esophageal cancer.

In another aspect, the invention pertains to a method of treatinggastric cancer comprising: selecting a patient suffering from gastriccancer; and administering an antibody or fragment thereof thatspecifically binds to a HER3 receptor, such that the antibody orfragment thereof binds to a conformational epitope comprising amino acidresidues within domain 2 and domain 4 of the HER3 receptor and blocksboth ligand-dependent and ligand-independent signal transduction,thereby treating gastric cancer.

In another aspect, the invention pertains to a method of treating headand neck cancer comprising selecting a patient suffering from cancer thehead and neck; and administering an antibody or fragment thereof thatspecifically binds to a HER3 receptor, such that the antibody orfragment thereof binds to a conformational epitope comprising amino acidresidues within domain 2 and domain 4 of the HER3 receptor and blocksboth ligand-dependent and ligand-independent signal transduction,thereby treating head and neck cancer.

In another aspect, the invention pertains to a method of treating benignprostate hypoplasia comprising: selecting a patient suffering frombenign prostate hypoplasia; and administering an antibody or fragmentthereof that specifically binds to a HER3 receptor, such that theantibody or fragment thereof binds to a conformational epitopecomprising amino acid residues within domain 2 and domain 4 of the HER3receptor and blocks both ligand-dependent and ligand-independent signaltransduction, thereby treating benign prostate hypoplasia.

In yet another aspect, the invention pertains to a method of treatinggynacomastica, comprising: selecting a patient suffering fromgynacomastica; and administering an antibody or fragment thereof thatspecifically binds to a HER3 receptor, such that the antibody orfragment thereof binds to a conformational epitope comprising amino acidresidues within domain 2 and domain 4 of the HER3 receptor and blocksboth ligand-dependent and ligand-independent signal transduction,thereby treating gynacomastica.

In yet another aspect, the invention pertains to a method of treatingendometriosis comprising: selecting a patient suffering fromendometriosis; and administering an antibody or fragment thereof thatspecifically binds to a HER3 receptor, such that the antibody orfragment thereof binds to a conformational epitope comprising amino acidresidues within domain 2 and domain 4 of the HER3 receptor and blocksboth ligand-dependent and ligand-independent signal transduction,thereby treating endometriosis.

In one embodiment, the antibody or fragment thereof is administered by aroute selected from the group consisting of oral, subcutaneous,intraperitoneal, intramuscular, intracerebroventricular,intraparenchymal, intrathecal, intracranial, buccal, mucosal, nasal, andrectal administration. In one embodiment, the antibody or fragmentthereof is formulated into a pharmaceutical composition comprising aphysiologically acceptable carrier, excipient, or diluent. In anotherembodiment, the pharmaceutical composition comprises an additionaltherapeutic agent. In one embodiment, the additional therapeutic agentis selected from the group consisting of an HER1 inhibitor, a HER2inhibitor, a HER3 inhibitor, a HER4 inhibitor, an mTOR inhibitor and aPI3 Kinase inhibitor. In one embodiment, the additional therapeuticagent is a HER1 inhibitor selected from the group consisting ofMatuzumab (EMD72000), Erbitux®/Cetuximab, Vectibix®/Panitumumab, mAb806, Nimotuzumab, Iressa®/Gefitinib, CI-1033 (PD183805), Lapatinib(GW-572016), Tykerb®/Lapatinib Ditosylate, Tarceva®/Erlotinib HCL(OSI-774), PKI-166, and Tovok®; a HER2 inhibitor selected from the groupconsisting of Pertuzumab, Trastuzumab, MM-111, neratinib, lapatinib orlapatinib ditosylate/Tykerb®; a HER3 inhibitor selected from the groupconsisting of, MM-121, MM-111, I1B4C3, 2DID12 (U3 Pharma AG), AMG888(Amgen), AV-203 (Aveo), MEHD7945A (Genentech), MORI0703 (Novartis) andsmall molecules that inhibit HER3; and a HER4 inhibitor. In oneembodiment, the additional therapeutic agent is an mTOR inhibitorselected from the group consisting of Temsirolimus/Torisel®,ridaforolimus/Deforolimus, AP23573, MK8669, everolimus/Affinitor®. Inone embodiment, the additional therapeutic agent is a PI3 Kinaseinhibitor selected from the group consisting of GDC 0941, BEZ235, BMK120and BYL719.

In another aspect, the invention pertains to use of an antibody orfragment thereof that specifically binds to a HER3 receptor, such thatthe antibody or fragment thereof binds to a conformational epitopecomprising amino acid residues within domain 2 and domain 4 of the HER3receptor and blocks both ligand-dependent and ligand-independent signaltransduction for the manufacture of a medicament for the treatment of anesophageal disorder, or gastric cancer, or head and neck cancer, orbenign prostatic hyperplasia (BPH), or gynacomastica, or endometriosis.

BRIEF DESCRIPTION OF FIGURES

FIG. 1: Representative MOR10701 SET curves obtained with human, mouse,rat and cyno HER3

FIG. 2: SK-Br-3 cell binding determination by FACS titration

FIG. 3: HER3 domain binding ELISA

FIGS. 4A-4C: Hydrogen deuterium exchange epitope mapping. A) HER3 ECDpeptides recovered following HDX-MS analysis are indicated by dashedlines. Potential N-linked glycosylation sites are highlighted. FIG. 4Adiscloses SEQ ID NO: 497. B) The relative degree of deuteration observedin peptides identified by MS. C) Protected residues mapped onto thepublished HER3 crystal structure.

FIGS. 5A-5E: A) Surface representation of the HER3/MOR09823 andHER3/MOR09825 x-ray crystal structures. HER3 (in lighter gray) is in theclosed conformation, and MOR09823 or MOR09825 (in darkest gray) bind toboth domains 2 and 4. B). Surface view of HER3 from the HER3/MOR09823structure shown in a similar orientation as (A). MOR09823 was omittedfor clarity. C) HER3/MOR09823 structure illustrated as a ribbonstructure, viewed at a 900 rotation from panels (A), (B) and (D). D) Aribbon representation of the inactive HER3 conformation recognized byMOR09823 Fab with a close up view of the domain 2/domain 4 interface,highlighting the HER3 residues (all positions disclosed in FIG. 5C areresidues of SEQ ID NO: 1) that are within 5 Å of the Fab. E) MutantHER3/MOR10703 binding determination by ELISA titration.

FIGS. 6A-6B: Inhibition of ligand induced (A) or ligand-independent (B)HER3 phosphorylation.

FIGS. 7A-7B: Inhibition of HER3 dependent downstream signaling pathwaysin HER2 amplified cell lines in (A) SKBR3 cells; and (B) in BT-474cells.

FIGS. 8A-8B: The impact of HER3 inhibition upon cell growth in A) BT-474and B) neuregulin stimulated MCF7 cells.

FIG. 9: The effect of MOR09823 and MOR09825 upon neuregulin binding toMCF7 cells.

FIG. 10: Impact of MOR09823 binding upon HER3/neuregulin complexformation as assessed by Biacore™. No antibody (black bars), MOR09823(white bars), 105.5 (grey) & control IgG (striped bars).

FIGS. 11A-11B: MOR09823 mediated inhibition of (A) ligand independent(BT-474) and (B) ligand dependent (BxPC3) HER3 signaling in vivo.

FIGS. 12A-12B: The impact of (A) MOR10701 and (B) MOR10703 upon BT-474tumor growth.

FIG. 13: The impact of MOR10701 and MOR10703 upon BxPC3 tumor growth.

FIGS. 14A-14H: MOR10703 in vitro drug combination isobolograms (A)MOR09823/trastuzumab, (B) MOR09823/lapatinib, (C) MOR10703/BEZ235, (D)MOR10703/BKM120, (E) MOR10703/BYL719, (F) MOR10703/RAD001, (G)MOR10703/cetuximab and (H) MOR10703/erlotinib.

FIGS. 15A-15B: MOR10701 or MOR10703 in vivo combinations with (A)trastuzumab and (B) erlotinib in BT-474 and L3.3.

FIG. 16: MOR10703 alone or MOR10703 in vitro combinations with cetuximabon esophageal cells

FIGS. 17A-17B: MOR10703 alone or MOR10703 in vivo combinations with (A)cetuximab and (B) BYL719 in KYSE140 and KYSE180 esophageal tumor models.

FIGS. 18A-18C: MOR10703 alone or MOR10703 in vivo combinations with (A)cetuximab and (B) BYL719 in CHE007 esophageal tumor models, and (C)MORI0703 alone or MOR10703 in vivo combinations with cetuximab inCHES015 esophageal tumor model.

FIG. 19: MOR10703 alone or MOR10703 in vivo combinations with BYL719 inN87 gastric tumor model showing prolonged tumor regression.

FIG. 20: MOR10703 alone or MOR10703 in vivo combinations with cetuximabin the A253 SCCHN model, treatment with either MOR10703 or cetuximab asa single agent resulted in tumor stasis. Combination of MOR10703 withcetuximab resulted in tumor regression.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The phrase “signal transduction” or “signaling activity” as used hereinrefers to a biochemical causal relationship generally initiated by aprotein-protein interaction such as binding of a growth factor to areceptor, resulting in transmission of a signal from one portion of acell to another portion of a cell. For HER3, the transmission involvesspecific phosphorylation of one or more tyrosine, serine, or threonineresidues on one or more proteins in the series of reactions causingsignal transduction. Penultimate processes typically include nuclearevents, resulting in a change in gene expression.

A “HER receptor” is a receptor protein tyrosine kinase which belongs tothe HER receptor family and includes EGFR, HER2, HER3 and HER4 receptorsand other members of this family to be identified in the future. The HERreceptor will generally comprise an extracellular domain, which may bindan HER ligand; a lipophilic transmembrane domain; a conservedintracellular tyrosine kinase domain; and a carboxyl-terminal signalingdomain harboring several tyrosine residues which can be phosphorylated.Preferably the HER receptor is native sequence human HER receptor.

The terms “HER1,” “ErbB1,” “epidermal growth factor receptor” and “EGFR”are used interchangeably herein and refer to EGFR as disclosed, forexample, in Carpenter et al. Ann. Rev. Biochem. 56:881-914 (1987),including naturally occurring mutant forms thereof (e.g. a deletionmutant EGFR as in Humphrey et al., (1990) PNAS (USA) 87:4207-4211).erbB1 refers to the gene encoding the EGFR protein product.

The terms “HER2” and “ErbB2” and are used interchangeably herein andrefer to human HER2 protein described, for example, in Semba et al.,(1985) PNAS (USA) 82:6497-6501 and Yamamoto et al. (1986) Nature319:230-234 (Genebank accession number X03363). The term “erbB2” refersto the gene encoding human ErbB2 and “neu” refers to the-gene encodingrat p85neu.

The terms “HER4” and “ErbB4” herein refer to the receptor polypeptide asdisclosed, for example, in EP Pat Appln No 599,274; Plowman et al.,(1993) Proc. Natl. Acad. Sci. USA, 90:1746-1750; and Plowman et al.,(1993) Nature, 366:473-475, including isoforms thereof, e.g., asdisclosed in WO99/19488, published Apr. 22, 1999.

The term “HER3” or “HER3 receptor” also known as “ErbB3” as used hereinrefers to mammalian HER3 protein and “her3” or “erbB3” refers tomammalian her3 gene. The preferred HER3 protein is human HER3 proteinpresent in the cell membrane of a cell. The human her3 gene is describedin U.S. Pat. No. 5,480,968 and Plowman et al., (1990) Proc. Natl. Acad.Sci. USA, 87:4905-4909.

Human HER3 as defined in Accession No. NP_001973 (human), andrepresented below as SEQ ID NO: 1. All nomenclature is for full length,immature HER3 (amino acids 1-1342). The immature HER3 is cleaved betweenpositions 19 and 20, resulting in the mature HER3 protein (20-1342 aminoacids).

(SEQ ID NO: 1) mrandalqvl gllfslargs evgnsqavcp gtlnglsvtgdaenqyqtly klyercevvm gnleivltgh nadlsflqwirevtgyvlva mnefstlplp nlrvvrgtqv ydgkfaifvmlnyntnssha lrqlrltqlt eilsggvyie kndklchmdtidwrdivrdr daeivvkdng rscppchevc kgrcwgpgsedcqtltktic apqcnghcfg pnpnqcchde caggcsgpqdtdcfacrhfn dsgacvprcp qplvynkltf qlepnphtkyqyggvcvasc phnfvvdqts cvracppdkm evdknglkmcepcgglcpka cegtgsgsrf qtvdssnidg fvnctkilgnldflitglng dpwhkipald peklnyfrtv reitgylniqswpphmhnfs vfsnlttigg rslynrgfsl limknlnvtslgfrslkeis agriyisanr qlcyhhslnw tkvlrgpteerldikhnrpr rdcvaegkvc dplcssggcw gpgpgqclscrnysrggvcv thcnflngep refaheaecf schpecqpmegtatcngsgs dtcaqcahfr dgphcvsscp hgvlgakgpiykypdvqnec rpchenctqg ckgpelqdcl gqtlvligkthltmaltvia glvvifmmlg gtflywrgrr iqnkramrrylergesiepl dpsekankvl arifketelr klkvlgsgvfgtvhkgvwip egesikipvc ikviedksgr qsfqavtdhmlaigsldhah ivrllglcpg sslqlvtqyl plgslldhvrqhrgalgpql llnwgvqiak gmyyleehgm vhrnlaarnvllkspsqvqv adfgvadllp pddkqllyse aktpikwmalesihfgkyth qsdvwsygvt vwelmtfgae pyaglrlaevpdllekgerl aqpqictidv ymvmvkcwmi denirptfkelaneftrmar dpprylvikr esgpgiapgp ephgltnkkleevelepeld ldldleaeed nlatttlgsa lslpygtlnrprgsqsllsp ssgympmnqg nlgescqesa vsgssercprpvslhpmprg clasessegh vtgseaelqe kvsmcrsrsrsrsprprgds ayhsqrhsll tpvtplsppg leeedvngyvmpdthlkgtp ssregtlssv glssylgtee ededeeyeymnrrrrhspph pprpssleel gyeymdvgsd lsaslgstqscplhpvpimp tagttpdedy eymnrqrdgg gpggdyaamgacpaseqgye emrafqgpgh qaphvhyarl ktlrsleatd safdnpdywh srlfpkanaq rt

The term “HER ligand” as used herein refers to polypeptides which bindand activate HER receptors such as HER1, HER2, HER3 and HER4. Examplesof HER ligands include, but are not limited to neuregulin 1 (NRG),neuregulin 2, neuregulin 3, neuregulin 4, betacellulin, heparin-bindingepidermal growth factor, epiregulin, epidermal growth factor,amphiregulin, and transforming growth factor alpha. The term includesbiologically active fragments and/or variants of a naturally occurringpolypeptide.

The term “HER3 ligand” as used herein refers to polypeptides which bindand activate HER3. Examples of HER3 ligands include, but are not limitedto neuregulin 1 (NRG) and neuregulin 2, betacellulin, heparin-bindingepidermal growth factor, and epiregulin. The term includes biologicallyactive fragments and/or variants of a naturally occurring polypeptide.

The “HER-HER protein complex” is a noncovalently associated oligomercontaining a HER co-receptors in any combination (e.g., HER1-HER2,HER1-HER3, HER-HER4, HER2-HER3, HER3-HER4, and the like). This complexcan form when a cell expressing both of these receptors is exposed to aHER ligand e.g., NRG, or when a HER receptor is active or overexpressed.

The “HER2-HER3 protein complex” is a noncovalently associated oligomercontaining HER2 receptor and the HER3 receptor. This complex can formwhen a cell expressing both of these receptors is exposed to a HER3ligand e.g., NRG or when HER2 is active/overexpressed. The phrase “HER3activity” or “HER3 activation” as used herein refers to an increase inoligomerization (e.g. an increase in HER3 containing complexes), HER3phosphorylation, conformational rearrangements (for example thoseinduced by ligands), and HER3 mediated downstream signaling.

The term “stabilization” or “stabilized” used in the context of HER3refers to an antibody or fragment thereof that directly maintains(locks, tethers, holds, preferentially binds, favors) the inactive stateor conformation of HER3 without blocking ligand binding to HER3, suchthat ligand binding is no longer able to activate HER3. Assays describedin the Examples can be used to measure ligand binding to a stabilizedHER3 receptor, e.g., Biacore assay.

The term “ligand-dependent signaling” as used herein refers to theactivation of HER (e.g., HER3) via ligand. HER3 activation is evidencedby increased oligomerization (e.g. heterodimerization) and/or HER3phosphorylation such that downstream signaling pathways (e.g. PI3K) areactivated. The antibody or fragment thereof can statisticallysignificantly reduce the amount of phosphorylated HER3 in a stimulatedcell exposed to the antigen binding protein (e.g., an antibody) relativeto an untreated (control) cell, as measured using the assays describedin the Examples. The cell which expresses HER3 can be a naturallyoccurring cell line (e.g. MCF7) or can be recombinantly produced byintroducing nucleic acids encoding HER3 protein into a host cell. Cellstimulation can occur either via the exogenous addition of an activatingHER3 ligand or by the endogenous expression of an activating ligand.

The antibody or fragment thereof which “reduces neregulin-induced HER3activation in a cell” is one which statistically significantly reducesHER3 tyrosine phosphorylation relative to an untreated (control) cell,as measured using the assays described in the Examples. This can bedetermined based on HER3 phosphotyrosine levels following exposure ofHER3 to NRG and the antibody of interest. The cell which expresses HER3protein can be a naturally occurring cell or cell line (e.g. MCF7) orcan be recombinantly produced.

The term “ligand-independent signaling” as used herein refers tocellular HER3 activity (e.g phosphorylation) in the absence of arequirement for ligand binding. For example, ligand-independent HER3activation can be a result of HER2 overexpression or activatingmutations in HER3 heterodimer partners such as EGFR and HER2. Theantibody or fragment thereof can statistically significantly reduce theamount of phosphorylated HER3 in a cell exposed to the antigen bindingprotein (e.g., an antibody) relative to an untreated (control) cell. Thecell which expresses HER3 can be a naturally occurring cell line (e.g.SK-Br-3) or can be recombinantly produced by introducing nucleic acidsencoding HER3 protein into a host cell.

The term “blocks” as used herein refers to stopping or preventing aninteraction or a process, e.g., stopping ligand-dependent orligand-independent signaling.

The term “recognize” as used herein refers to an antibody or fragmentthereof that finds and interacts (e.g., binds) with its conformationalepitope.

The phrase “concurrently binds” as used herein refers to a HER ligandthat can bind to a ligand binding site on the HER receptor along withthe HER antibody. This means that both the antibody and antibody canbind to the HER receptor together. For the sake of illustration only,the HER3 ligand NRG, can bind to the HER3 receptor along with the HER3antibody. Assay to measure concurrent binding of the ligand and antibodyare described in the Examples section (e.g., Biacore).

The term “fails” as used herein refers to an antibody or fragmentthereof that does not do a particular event. For example, an antibody orfragment thereof that “fails to activate signal transduction” is onethat does not trigger signal transduction; an antibody or fragmentthereof that “fails to induce a conformational change” is one that doesnot cause a structural alteration in the HER receptor; an antibody orfragment thereof that stabilizes the HER receptor in an inactive statesuch that the HER receptor “fails to dimerize” is one that does not formprotein-protein complexes.

The term “antibody” as used herein refers to whole antibodies thatinteract with (e.g., by binding, steric hindrance,stabilizing/destabilizing, spatial distribution) an HER3 epitope andinhibit signal transduction. A naturally occurring “antibody” is aglycoprotein comprising at least two heavy (H) chains and two light (L)chains inter-connected by disulfide bonds. Each heavy chain is comprisedof a heavy chain variable region (abbreviated herein as VH) and a heavychain constant region. The heavy chain constant region is comprised ofthree domains, CH1, CH2 and CH3. Each light chain is comprised of alight chain variable region (abbreviated herein as VL) and a light chainconstant region. The light chain constant region is comprised of onedomain, CL. The VH and VL regions can be further subdivided into regionsof hypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each VH and VL is composed of three CDRs and four FRsarranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavyand light chains contain a binding domain that interacts with anantigen. The constant regions of the antibodies may mediate the bindingof the immunoglobulin to host tissues or factors, including variouscells of the immune system (e.g., effector cells) and the firstcomponent (C1q) of the classical complement system. The term “antibody”includes for example, monoclonal antibodies, human antibodies, humanizedantibodies, camelised antibodies, chimeric antibodies, single-chain Fvs(scFv), disulfide-linked Fvs (sdFv), Fab fragments, F (ab′) fragments,and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Idantibodies to antibodies of the invention), and epitope-bindingfragments of any of the above. The antibodies can be of any isotype(e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3,IgG4, IgA1 and IgA2) or subclass.

Both the light and heavy chains are divided into regions of structuraland functional homology. The terms “constant” and “variable” are usedfunctionally. In this regard, it will be appreciated that the variabledomains of both the light (VL) and heavy (VH) chain portions determineantigen recognition and specificity. Conversely, the constant domains ofthe light chain (CL) and the heavy chain (CH1, CH2 or CH3) conferimportant biological properties such as secretion, transplacentalmobility, Fc receptor binding, complement binding, and the like. Byconvention the numbering of the constant region domains increases asthey become more distal from the antigen binding site or amino-terminusof the antibody. The N-terminus is a variable region and at theC-terminus is a constant region; the CH3 and CL domains actuallycomprise the carboxy-terminus of the heavy and light chain,respectively.

The phrase “antibody fragment”, as used herein, refers to one or moreportions of an antibody that retain the ability to specifically interactwith (e.g., by binding, steric hindrance, stabilizing/destabilizing,spatial distribution) an HER3 epitope and inhibit signal transduction.Examples of binding fragments include, but are not limited to, a Fabfragment, a monovalent fragment consisting of the VL, VH, CL and CH1domains; a F(ab)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; a Fdfragment consisting of the VH and CH1 domains; a Fv fragment consistingof the VL and VH domains of a single arm of an antibody; a dAb fragment(Ward et al., (1989) Nature 341:544-546), which consists of a VH domain;and an isolated complementarity determining region (CDR).

Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they can be joined, using recombinantmethods, by a synthetic linker that enables them to be made as a singleprotein chain in which the VL and VH regions pair to form monovalentmolecules (known as single chain Fv (scFv); see e.g., Bird et al.,(1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad.Sci. 85:5879-5883). Such single chain antibodies are also intended to beencompassed within the term “antibody fragment”. These antibodyfragments are obtained using conventional techniques known to those ofskill in the art, and the fragments are screened for utility in the samemanner as are intact antibodies.

Antibody fragments can also be incorporated into single domainantibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies,tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, (2005)Nature Biotechnology 23:1126-1136). Antibody fragments can be graftedinto scaffolds based on polypeptides such as Fibronectin type III (Fn3)(see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptidemonobodies).

Antibody fragments can be incorporated into single chain moleculescomprising a pair of tandem Fv segments (VH-CH1-VH-CH1) which, togetherwith complementary light chain polypeptides, form a pair of antigenbinding regions (Zapata et al., (1995) Protein Eng. 8:1057-1062; andU.S. Pat. No. 5,641,870).

The term “epitope” includes any protein determinant capable of specificbinding to an immunoglobulin or otherwise interacting with a molecule.Epitopic determinants generally consist of chemically active surfacegroupings of molecules such as amino acids or carbohydrate or sugar sidechains and can have specific three-dimensional structuralcharacteristics, as well as specific charge characteristics. An epitopemay be “linear” or “conformational.”

The term “linear epitope” refers to an epitope with all of the points ofinteraction between the protein and the interacting molecule (such as anantibody) occur linearly along the primary amino acid sequence of theprotein (continuous). Once a desired epitope on an antigen isdetermined, it is possible to generate antibodies to that epitope, e.g.,using the techniques described in the present invention. Alternatively,during the discovery process, the generation and characterization ofantibodies may elucidate information about desirable epitopes. From thisinformation, it is then possible to competitively screen antibodies forbinding to the same epitope. An approach to achieve this is to conductcross-competition studies to find antibodies that competitively bindwith one another, e.g., the antibodies compete for binding to theantigen. A high throughput process for “binning” antibodies based upontheir cross-competition is described in International Patent ApplicationNo. WO 2003/48731. As will be appreciated by one of skill in the art,practically anything to which an antibody can specifically bind could bean epitope. An epitope can comprises those residues to which theantibody binds.

The term “conformational epitope” refers to an epitope in whichdiscontinuous amino acids that come together in three dimensionalconformation. In a conformational epitope, the points of interactionoccur across amino acid residues on the protein that are separated fromone another. In one embodiment, the epitope is that described inExamples of this specification. In one embodiment, the conformationalepitope is defined by (i) HER3 amino acid residues 265-277 and 315 (ofdomain 2) and (ii) HER3 amino acid residues 571, 582-584, 596-597,600-602, 609-615 (of domain 4) of SEQ ID NO: 1, or a subset thereof. Aswill be appreciated by one of skill in the art, the space that isoccupied by a residue or side chain that creates the shape of a moleculehelps to determine what an epitope is.

Generally, antibodies specific for a particular target antigen willpreferentially recognize an epitope on the target antigen in a complexmixture of proteins and/or macromolecules.

Regions of a given polypeptide that include an epitope can be identifiedusing any number of epitope mapping techniques, well known in the art.See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology,Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, N.J. Forexample, linear epitopes may be determined by e.g., concurrentlysynthesizing large numbers of peptides on solid supports, the peptidescorresponding to portions of the protein molecule, and reacting thepeptides with antibodies while the peptides are still attached to thesupports. Such techniques are known in the art and described in, e.g.,U.S. Pat. No. 4,708,871; Geysen et al., (1984) Proc. Natl. Acad. Sci.USA 8:3998-4002; Geysen et al., (1985) Proc. Natl. Acad. Sci. USA82:78-182; Geysen et al., (1986) Mol. Immunol. 23:709-715. Similarly,conformational epitopes are readily identified by determining spatialconformation of amino acids such as by, e.g., hydrogen/deuteriumexchange, x-ray crystallography and two-dimensional nuclear magneticresonance. See, e.g., Epitope Mapping Protocols, supra. Antigenicregions of proteins can also be identified using standard antigenicityand hydropathy plots, such as those calculated using, e.g., the Omigaversion 1.0 software program available from the Oxford Molecular Group.This computer program employs the Hopp/Woods method, Hopp et al., (1981)Proc. Natl. Acad. Sci USA 78:3824-3828; for determining antigenicityprofiles, and the Kyte-Doolittle technique, Kyte et al., (1982) J. Mol.Biol. 157:105-132; for hydropathy plots.

The term “paratope” as used herein refers to the general structure of abinding region that determines binding to an epitope. This structureinfluences whether or not and in what manner the binding region mightbind to an epitope. Paratope can refer to an antigenic site of anantibody that is responsible for an antibody or fragment thereof, tobind to an antigenic determinant. Paratope also refers to the idiotopeof the antibody, and the complementary determining region (CDR) regionthat binds to the epitope. In one embodiment, the paratope is the regionof the antibody that binds to the conformational epitope comprising (i)HER3 amino acid residues 265-277 and 315 (of domain 2), and (ii) HER3amino acid residues 571, 582-584, 596-597, 600-602, 609-615 (of domain4) of SEQ ID NO: 1, or a subset thereof. In one embodiment, the paratopeis the region of the antibody that comprises the CDR sequences. In oneembodiment, the paratope comprises the sequences listed in Table 1. Inone embodiment, the paratope comprises at least one amino acid residuethat binds with HER3 residues: Asn266, Lys267, Leu268, Thr269, Gln271,Glu273, Pro274, Asn275, Pro276, His277, Asn315, Asp571, Pro583, His584,Ala596, Lys597 (residues of SEQ ID NO: 1). In one embodiment, theparatope comprises at least one amino acid residue that binds with HER3residues: Tyr265, Lys267, Leu268, Phe270, Gly582, Pro583, Lys597,Ile600, Lys602, Glu609, Arg611, Pro612, Cys613, His614, Glu615 (residuesof SEQ ID NO: 1). As will be appreciated by one of skill in the art, theparatope of any antibody, or variant thereof, can be determined in themanner set forth by the present application.

The phrases “monoclonal antibody” or “monoclonal antibody composition”as used herein refers to polypeptides, including antibodies, antibodyfragments, bispecific antibodies, etc. that have substantially identicalto amino acid sequence or are derived from the same genetic source. Thisterm also includes preparations of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.

The phrase “human antibody”, as used herein, includes antibodies havingvariable regions in which both the framework and CDR regions are derivedfrom sequences of human origin. Furthermore, if the antibody contains aconstant region, the constant region also is derived from such humansequences, e.g., human germline sequences, or mutated versions of humangermline sequences or antibody containing consensus framework sequencesderived from human framework sequences analysis, for example, asdescribed in Knappik et al., (2000) J Mol Biol 296:57-86). Thestructures and locations of immunoglobulin variable domains, e.g., CDRs,may be defined using well known numbering schemes, e.g., the Kabatnumbering scheme, the Chothia numbering scheme, or a combination ofKabat and Chothia (see, e.g., Sequences of Proteins of ImmunologicalInterest, U.S. Department of Health and Human Services (1991), eds.Kabat et al.; Lazikani et al., (1997) J. Mol. Bio. 273:927-948); Kabatet al., (1991) Sequences of Proteins of Immunological Interest, 5thedit., NIH Publication no. 91-3242 U.S. Department of Health and HumanServices; Chothia et al., (1987) J. Mol. Biol. 196:901-917; Chothia etal., (1989) Nature 342:877-883; and Al-Lazikani et al., (1997) J. Mol.Biol. 273:927-948.

The human antibodies of the invention may include amino acid residuesnot encoded by human sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo, or aconservative substitution to promote stability or manufacturing).

The phrase “human monoclonal antibody” as used herein refers toantibodies displaying a single binding specificity which have variableregions in which both the framework and CDR regions are derived fromhuman sequences. In one embodiment, the human monoclonal antibodies areproduced by a hybridoma which includes a B cell obtained from atransgenic nonhuman animal, e.g., a transgenic mouse, having a genomecomprising a human heavy chain transgene and a light chain transgenefused to an immortalized cell.

The phrase “recombinant human antibody”, as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as antibodies isolated from an animal (e.g., amouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom, antibodies isolated from a hostcell transformed to express the human antibody, e.g., from atransfectoma, antibodies isolated from a recombinant, combinatorialhuman antibody library, and antibodies prepared, expressed, created orisolated by any other means that involve splicing of all or a portion ofa human immunoglobulin gene, sequences to other DNA sequences. Suchrecombinant human antibodies have variable regions in which theframework and CDR regions are derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the VH and VL regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline VH and VL sequences, may not naturally existwithin the human antibody germline repertoire in vivo.

Specific binding between two entities means a binding with anequilibrium constant (K_(A)) (k_(on)/k_(off)) of at least 10²M⁻¹, atleast 5×10²M⁻¹, at least 10³M⁻¹, at least 5×10³M⁻¹, at least 10⁴M⁻¹ atleast 5×10⁴M⁻¹, at least 10⁵M⁻¹, at least 5×10⁵M⁻¹, at least 10⁶M⁻¹, atleast 5×10⁶M⁻¹, at least 10⁷M⁻¹, at least 5×10⁷M⁻¹, at least 10⁸M⁻¹, atleast 5×10⁸M⁻¹, at least 10⁹M⁻¹, at least 5×10⁹M⁻¹, at least 10¹⁰M⁻¹, atleast 5×10¹⁰M⁻¹, at least 10¹¹M⁻¹, at least 5×10¹¹M⁻¹, at least 10¹²M⁻¹,at least 5×10¹²M⁻¹, at least 10¹³M⁻¹, at least 5×10¹³ M⁻¹, at least10¹⁴M⁻¹, at least 5×10¹⁴M⁻¹, at least 10¹⁵M⁻¹, or at least 5×10¹⁵M⁻¹.

The phrase “specifically (or selectively) binds” to an antibody (e.g., aHER3 binding antibody) refers to a binding reaction that isdeterminative of the presence of a cognate antigen (e.g., a human HER3)in a heterogeneous population of proteins and other biologics. Inaddition to the equilibrium constant (K_(A)) noted above, an HER3binding antibody of the invention typically also has a dissociation rateconstant (K_(D)) (k_(off)/k_(on)) of less than 5×10⁻²M, less than 10⁻²M,less than 5×10⁻³M, less than 10⁻³M, less than 5×10⁻⁴M, less than 10⁴M,less than 5×10⁻⁵M, less than 10⁻⁵M, less than 5×10⁻⁶M, less than 10⁻⁶M,less than 5×10⁻⁷M, less than 10⁻⁷M, less than 5×10⁻⁸M, less than 10⁻⁸M,less than 5×10⁻⁹M, less than 10⁻⁹M, less than 5×10⁻¹⁰M, less than10⁻¹⁰M, less than 5×10⁻¹¹M, less than 10⁻¹¹M, less than 5×10⁻¹²M, lessthan 10⁻¹²M, less than 5×10⁻¹³M, less than 10⁻¹³M, less than 5×10⁻¹⁴M,less than 10⁻¹⁴M, less than 5×10⁻¹⁵M, or less than 10⁻¹⁵M or lower, andbinds to HER3 with an affinity that is at least two-fold greater thanits affinity for binding to a non-specific antigen (e.g., HSA).

In one embodiment, the antibody or fragment thereof has dissociationconstant (K_(d)) of less than 3000 pM, less than 2500 pM, less than 2000pM, less than 1500 pM, less than 1000 pM, less than 750 pM, less than500 pM, less than 250 pM, less than 200 pM, less than 150 pM, less than100 pM, less than 75 pM, less than 10 pM, less than 1 pM as assessedusing a method described herein or known to one of skill in the art(e.g., a BIAcore assay, ELISA, FACS, SET) (Biacore International AB,Uppsala, Sweden). The term “K_(assoc)” or “K_(a)”, as used herein,refers to the association rate of a particular antibody-antigeninteraction, whereas the term “K_(dis)” or “K_(d),” as used herein,refers to the dissociation rate of a particular antibody-antigeninteraction. The term “K_(D)”, as used herein, refers to thedissociation constant, which is obtained from the ratio of K_(d) toK_(a) (i.e. K_(d)/K_(a)) and is expressed as a molar concentration (M).K_(D) values for antibodies can be determined using methods wellestablished in the art. A method for determining the K_(D) of anantibody is by using surface plasmon resonance, or using a biosensorsystem such as a Biacore® system.

The term “affinity” as used herein refers to the strength of interactionbetween antibody and antigen at single antigenic sites. Within eachantigenic site, the variable region of the antibody “arm” interactsthrough weak non-covalent forces with antigen at numerous sites; themore interactions, the stronger the affinity.

The term “avidity” as used herein refers to an informative measure ofthe overall stability or strength of the antibody-antigen complex. It iscontrolled by three major factors: antibody epitope affinity; thevalence of both the antigen and antibody; and the structural arrangementof the interacting parts. Ultimately these factors define thespecificity of the antibody, that is, the likelihood that the particularantibody is binding to a precise antigen epitope.

The term “valency” as used herein refers to the number of potentialtarget binding sites in a polypeptide. Each target binding sitespecifically binds one target molecule or specific site (i.e, epitope)on a target molecule. When a polypeptide comprises more than one targetbinding site, each target binding site may specifically bind the same ordifferent molecules (e.g., may bind to different molecules, e.g.,different antigens, or different epitopes on the same molecule).

The phrase “antagonist antibody” as used herein refers to an antibodythat binds with HER3 and neutralizes the biological activity of HER3signaling, e.g., reduces, decreases and/or inhibits HER3 inducedsignaling activity, e.g., in a phospho-HER3 or phospho-Akt assay.Examples of assays are described in more details in the examples below.Accordingly, an antibody that “inhibits” one or more of these HER3functional properties (e.g., biochemical, immunochemical, cellular,physiological or other biological activities, or the like) as determinedaccording to methodologies known to the art and described herein, willbe understood to relate to a statistically significant decrease in theparticular activity relative to that seen in the absence of the antibody(e.g., or when a control antibody of irrelevant specificity is present).An antibody that inhibits HER3 activity effects such a statisticallysignificant decrease by at least 10% of the measured parameter, by atleast 50%, 80% or 90%, and in certain embodiments an antibody of theinvention may inhibit greater than 95%, 98% or 99% of HER3 functionalactivity as evidenced by a reduction in the level of cellular HER3phosphorylation.

The phrase “isolated antibody” refers to an antibody that issubstantially free of other antibodies having different antigenicspecificities (e.g., an isolated antibody that specifically binds HER3is substantially free of antibodies that specifically bind antigensother than HER3). An isolated antibody that specifically binds HER3 may,however, have cross-reactivity to other antigens. Moreover, an isolatedantibody may be substantially free of other cellular material and/orchemicals.

The phrase “conservatively modified variant” applies to both amino acidand nucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidthat encodes a polypeptide is implicit in each described sequence.

For polypeptide sequences, “conservatively modified variants” includeindividual substitutions, deletions or additions to a polypeptidesequence which result in the substitution of an amino acid with achemically similar amino acid. Conservative substitution tablesproviding functionally similar amino acids are well known in the art.Such conservatively modified variants are in addition to and do notexclude polymorphic variants, interspecies homologs, and alleles of theinvention. The following eight groups contain amino acids that areconservative substitutions for one another: 1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins (1984)). In someembodiments, the term “conservative sequence modifications” are used torefer to amino acid modifications that do not significantly affect oralter the binding characteristics of the antibody containing the aminoacid sequence.

The terms “cross-compete” and “cross-competing” are used interchangeablyherein to mean the ability of an antibody or other binding agent tointerfere with the binding of other antibodies or binding agents to HER3in a standard competitive binding assay.

The ability or extent to which an antibody or other binding agent isable to interfere with the binding of another antibody or bindingmolecule to HER3, and therefore whether it can be said to cross-competeaccording to the invention, can be determined using standard competitionbinding assays. One suitable assay involves the use of the Biacoretechnology (e.g. by using the BIAcore 3000 instrument (Biacore, Uppsala,Sweden)), which can measure the extent of interactions using surfaceplasmon resonance technology. Another assay for measuringcross-competing uses an ELISA-based approach.

The term “optimized” as used herein refers to a nucleotide sequence hasbeen altered to encode an amino acid sequence using codons that arepreferred in the production cell or organism, generally a eukaryoticcell, for example, a cell of Pichia, a cell of Trichoderma, a ChineseHamster Ovary cell (CHO) or a human cell. The optimized nucleotidesequence is engineered to retain completely or as much as possible theamino acid sequence originally encoded by the starting nucleotidesequence, which is also known as the “parental” sequence.

Standard assays to evaluate the binding ability of the antibodies towardHER3 of various species are known in the art, including for example,ELISAs, western blots and RIAs. Suitable assays are described in detailin the Examples. The binding kinetics (e.g., binding affinity) of theantibodies also can be assessed by standard assays known in the art,such as by Biacore analysis, or FACS relative affinity (Scatchard).Assays to evaluate the effects of the antibodies on functionalproperties of HER3 (e.g., receptor binding assays, modulating the Herpathway) are described in further detail in the Examples.

The phrases “percent identical” or “percent identity,” in the context oftwo or more nucleic acids or polypeptide sequences, refers to two ormore sequences or subsequences that are the same. Two sequences are“substantially identical” if two sequences have a specified percentageof amino acid residues or nucleotides that are the same (i.e., 60%identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identityover a specified region, or, when not specified, over the entiresequence), when compared and aligned for maximum correspondence over acomparison window, or designated region as measured using one of thefollowing sequence comparison algorithms or by manual alignment andvisual inspection. Optionally, the identity exists over a region that isat least about 50 nucleotides (or 10 amino acids) in length, or morepreferably over a region that is 100 to 500 or 1000 or more nucleotides(or 20, 50, 200 or more amino acids) in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith and Waterman, (1970) Adv. Appl. Math. 2:482c, by the homologyalignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol.48:443, by the search for similarity method of Pearson and Lipman,(1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by manual alignment and visualinspection (see, e.g., Brent et al., (2003) Current Protocols inMolecular Biology).

Two examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al., (1977) Nuc. AcidsRes. 25:3389-3402; and Altschul et al., (1990) J. Mol. Biol.215:403-410, respectively. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation. This algorithm involves first identifying high scoringsequence pairs (HSPs) by identifying short words of length W in thequery sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al., supra). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are extended in both directions along each sequencefor as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) or 10, M=5, N=−4 and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and acomparison of both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul, (1993)Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

The percent identity between two amino acid sequences can also bedetermined using the algorithm of E. Meyers and W. Miller, (1988)Comput. Appl. Biosci. 4:11-17) which has been incorporated into theALIGN program (version 2.0), using a PAM120 weight residue table, a gaplength penalty of 12 and a gap penalty of 4. In addition, the percentidentity between two amino acid sequences can be determined using theNeedleman and Wunsch (1970) J. Mol. Biol. 48:444-453) algorithm whichhas been incorporated into the GAP program in the GCG software package(available at www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6.

Other than percentage of sequence identity noted above, anotherindication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by conservative substitutions. Another indication that twonucleic acid sequences are substantially identical is that the twomolecules or their complements hybridize to each other under stringentconditions, as described below. Yet another indication that two nucleicacid sequences are substantially identical is that the same primers canbe used to amplify the sequence.

The phrase “nucleic acid” is used herein interchangeably with the term“polynucleotide” and refers to deoxyribonucleotides or ribonucleotidesand polymers thereof in either single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, as detailed below,degenerate codon substitutions may be achieved by generating sequencesin which the third position of one or more selected (or all) codons issubstituted with mixed-base and/or deoxyinosine residues (Batzer et al.,(1991) Nucleic Acid Res. 19:5081; Ohtsuka et al., (1985) J. Biol. Chem.260:2605-2608; and Rossolini et al., (1994) Mol. Cell. Probes 8:91-98).

The phrase “operably linked” refers to a functional relationship betweentwo or more polynucleotide (e.g., DNA) segments. Typically, it refers tothe functional relationship of a transcriptional regulatory sequence toa transcribed sequence. For example, a promoter or enhancer sequence isoperably linked to a coding sequence if it stimulates or modulates thetranscription of the coding sequence in an appropriate host cell orother expression system. Generally, promoter transcriptional regulatorysequences that are operably linked to a transcribed sequence arephysically contiguous to the transcribed sequence, i.e., they arecis-acting. However, some transcriptional regulatory sequences, such asenhancers, need not be physically contiguous or located in closeproximity to the coding sequences whose transcription they enhance.

The terms “polypeptide” and “protein” are used interchangeably herein torefer to a polymer of amino acid residues. The terms apply to amino acidpolymers in which one or more amino acid residue is an artificialchemical mimetic of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid polymers and non-naturallyoccurring amino acid polymer. Unless otherwise indicated, a particularpolypeptide sequence also implicitly encompasses conservatively modifiedvariants thereof.

The term “subject” includes human and non-human animals. Non-humananimals include all vertebrates, e.g., mammals and non-mammals, such asnon-human primates, sheep, dog, cow, chickens, amphibians, and reptiles.Except when noted, the terms “patient” or “subject” are used hereininterchangeably.

The term “anti-cancer agent” means any agent that can be used to treat acell proliferative disorder such as cancer, including cytotoxic agents,chemotherapeutic agents, radiotherapy and radiotherapeutic agents,targeted anti-cancer agents, and immunotherapeutic agents.

“Tumor” refers to neoplastic cell growth and proliferation, whethermalignant or benign, and all pre-cancerous and cancerous cells andtissues.

The term “anti-tumor activity” means a reduction in the rate of tumorcell proliferation, viability, or metastatic activity. A possible way ofshowing anti-tumor activity is show a decline in growth rate of abnormalcells that arises during therapy or tumor size stability or reduction.Such activity can be assessed using accepted in vitro or in vivo tumormodels, including but not limited to xenograft models, allograft models,MMTV models, and other known models known in the art to investigateanti-tumor activity.

The term “malignancy” refers to a non-benign tumor or a cancer. As usedherein, the term “cancer” includes a malignancy characterized byderegulated or uncontrolled cell growth. Exemplary cancers include:carcinomas, sarcomas, leukemias, and lymphomas. The term “cancer”includes primary malignant tumors (e.g., those whose cells have notmigrated to sites in the subject's body other than the site of theoriginal tumor) and secondary malignant tumors (e.g., those arising frommetastasis, the migration of tumor cells to secondary sites that aredifferent from the site of the original tumor).

Various aspects of the invention are described in further detail in thefollowing sections and subsections.

Structure and Mechanism of Activation of the HER Receptors

All four HER receptors have an extracellular ligand-binding domain, asingle trans-membrane domain and a cytoplasmic tyrosinekinase-containing domain. The intracellular tyrosine kinase domain ofHER receptors is highly conserved, although the kinase domain of HER3contains substitutions of critical amino acids and therefore lackskinase activity (Guy et al., (1994): PNAS 91, 8132-8136). Ligand-induceddimerisation of the HER receptors induces activation of the kinase,receptor transphosphorylation on tyrosine residues in the C-terminaltail, followed by recruitment and activation of intracellular signallingeffectors (Yarden and Sliwkowski, (2001) Nature Rev 2, 127-137; Jorissenet al., (2003) Exp Cell Res 284, 31-53.

The crystal structures of the extracellular domains of HERs haveprovided some insight into the process of ligand-induced receptoractivation (Schlessinger, (2002) Cell 110, 669-672). The extracellulardomain of each HER receptor consists of four subdomains: Subdomain I andIII cooperate in forming the ligand-binding site, whereas subdomain II(and perhaps also subdomain IV) participates in receptor dimerisationvia direct receptor-receptor interactions. In the structures ofligand-bound HER1, a hairpin (termed the dimerisation loop) in subdomainII interacts with the dimerisation loop of the partner receptor,mediating receptor dimerisation (Garrett et al, (2002) Cell 110,763-773; Ogiso et al., (2002) Cell 110, 775-787). In contrast, in thestructures of the inactive HER1, HER3 and HER4, the dimerisation loop isengaged in intramolecular interactions with subdomain IV, which preventsreceptor dimerisation in the absence of ligand (Cho and Leahy, (2002)Science 297, 1330-1333; Ferguson et al., (2003) Mol Cell 12, 541-552;Bouyan et al., (2005) PNAS102, 15024-15029). The structure of HER2 isunique among the HERs. In the absence of a ligand, HER2 has aconformation that resembles the ligand-activated state of HER1 with aprotruding dimerisation loop, available to interact with other HERreceptors (Cho et al., (2003) Nature 421, 756-760; Garrett et al.,(2003) Mol Cell 11, 495-505). This may explain the enhancedheterodimerisation capacity of HER2.

Although the HER receptor crystal structures provide a model for HERreceptor homo- and heterodimerisation, the background for the prevalenceof some HER homo- and heterodimers over others (Franklin et al., (2004)Cancer Cell 5, 317-328) as well as the conformational role of each ofthe domain in receptor dimerisation and autoinhibition (Burgess et al.,(2003) Mol Cell 12, 541-552; Mattoon et al., (2004) PNAS101, 923-928)remains somewhat unclear. As described below, the HER3 X-ray crystalstructure provides more insights.

HER3 Structure and Conformational Epitopes

A conformational epitope to which antigen binding proteins, e.g.,anti-HER3 antibodies bind is provided herein. For the first time, thethree dimensional structure of a truncated form (residues 20-640) of theextracellular domain of HER3 complexed with an antibody have been shown.The HER3-MOR09823 Fab complex and the HER3-MOR09825 have been determinedat 3.2 Å and 3.4 Å resolution, respectively, and shown in FIG. 5A. Thedisclosure herein also shows for the first time an antibody or fragmentthereof that binds to an inactive state of HER3 and stabilizes thereceptor in the inactive state. The antibodies of the invention alsopermit concurrent binding of a HER3 ligand, such as neuregulin with theHER3 receptor.

Although not bound to provide a theory, one possible model for themechanism of action is that HER3 typically exists in an inactive(closed, tethered) or active (open) state. Ligand binding induces aconformational change such that HER3 exists in the active (open) statewhich is capable of binding heterodimer partners resulting in activationin downstream signaling. Antibodies such as MOR09823 bind the inactive(tethered) state of HER3 but do not block the ligand binding site.Antibodies such as MOR09823 inhibit HER3 by preventing the ligandinduced structural rearrangements required for HER3 to transition to theactive conformation, thereby preventing signal transduction. In oneembodiment, the antibodies of the invention or fragments thereof bindthe inactive (tethered) state of HER3 but do not block the ligandbinding site. In another embodiment, the antibodies or fragments thereofinhibit HER3 by preventing the ligand-induced structural rearrangementsrequired for HER3 to transition to the active conformation, therebypreventing signal transduction. In another embodiment, the antibody orfragment thereof stabilizes (directly maintains, locks, tethers, holds,preferentially binds, or favors) HER3 receptor in the inactive state orconformation. In one embodiment, the inactive HER3 receptor may besusceptible to preferential internalization or degradation such that itleads to loss of cell surface HER3 receptors. The biological datapresented in the Examples section supports these embodiments. Thecrystals of HER3 may be prepared by expressing a nucleotide sequenceencoding HER3 or a variant thereof in a suitable host cell, and thencrystallising the purified protein(s) in the presence of the relevantHER3 targeted Fab. Preferably the HER3 polypeptide contains theextracellular domain (amino acids 20 to 640 of the human polypeptide ora truncated version thereof, preferably comprising amino acids 20-640)but lacks the transmembrane and intracellular domains.

HER3 polypeptides may also be produced as fusion proteins, for exampleto aid in extraction and purification. Examples of fusion proteinpartners include glutathione-S-transferase (GST), histidine (HIS),hexahistidine (6HIS) (SEQ ID NO: 495), GAL4 (DNA binding and/ortranscriptional activation domains) and beta-galactosidase. It may alsobe convenient to include a proteolytic cleavage site between the fusionprotein partner and the protein sequence of interest to allow removal offusion protein sequences.

After expression, the proteins may be purified and/or concentrated, forexample by immobilised metal affinity chromatography, ion-exchangechromatography, and/or gel filtration.

The protein(s) may be crystallised using techniques described herein.Commonly, in a crystallisation process, a drop containing the proteinsolution is mixed with the crystallisation buffer and allowed toequilibrate in a sealed container. Equilibration may be achieved byknown techniques such as the “hanging drop” or the “sitting drop”method. In these methods, the drop is hung above or sitting beside amuch larger reservoir of crystallization buffer and equilibration isreached through vapor diffusion. Alternatively, equilibration may occurby other methods, for example under oil, through a semi-permeablemembrane, or by free-interface diffusion (See e.g., Chayen et al.,(2008) Nature Methods 5, 147-153.

Once the crystals have been obtained, the structure may be solved byknown X-ray diffraction techniques. Many techniques use chemicallymodified crystals, such as those modified by heavy atom derivatizationto approximate phases. In practice, a crystal is soaked in a solutioncontaining heavy metal atom salts, or organometallic compounds, e.g.,lead chloride, gold thiomalate, thimerosal or uranyl acetate, which candiffuse through the crystal and bind to the surface of the protein. Thelocation(s) of the bound heavy metal atom(s) can then be determined byX-ray diffraction analysis of the soaked crystal. The patterns obtainedon diffraction of a monochromatic beam of X-rays by the atoms(scattering centres) of the crystal can be solved by mathematicalequations to give mathematical coordinates. The diffraction data areused to calculate an electron density map of the repeating unit of thecrystal. Another method of obtaining phase information is using atechnique known as molecular replacement. In this method, rotational andtranslational algorithms are applied to a search model derived from arelated structure, resulting in an approximate orientation for theprotein of interest (See Rossmann, (1990) Acta Crystals A 46, 73-82).The electron density maps are used to establish the positions of theindividual atoms within the unit cell of the crystal (Blundel et al.,(1976) Protein Crystallography, Academic Press).

The present disclosure describes for the first time, thethree-dimensional structure of HER3 and a Fab of an anti-HER3 antibody.The approximate domain boundaries of extracellular domain of HER3 are asfollows; domain 1: amino acids 20-207; domain 2: amino acids 208-328;domain 3: amino acids 329-498; and domain 4: amino acids 499-642. Thethree-dimensional structure of HER3 and the antibody also allows theidentification of target binding sites for potential HER3 modulators.Preferred target binding sites are those involved in the activation ofHER3. In one embodiment, the target binding site is located withindomain 2 and domain 4 of HER3. Thus an antibody or fragment thereofwhich binds to either domain 2 or domain 4, and preferably to bothdomains can modulate HER3 activation by either preventing the domainsfrom dissociation from each other or by modifying the relative positionsof the domains. Thus binding an antibody or fragment thereof to aminoacid residues within domain 2 or domain 4 may cause the protein to adopta conformation that prevents activation. The disclosure herein alsoshows for the first time an antibody or fragment thereof that canconcurrently bind with a HER3 ligand, such as neuregulin.

In some embodiments, the antibody or fragment thereof recognize aspecific conformational state of HER3 such that the antibody or fragmentthereof prevents HER3 from interacting with a co-receptor (including,but not limited to, HER1, HER2 and HER4). In some embodiments, theantibody or fragment thereof prevents HER3 from interacting with aco-receptor by stabilizing the HER3 receptor in an inactive or closedstate. In one embodiment, the antibody or fragment thereof stabilizesthe HER3 receptor by binding to amino acid residues within domain 2 anddomain 4 of HER3. In this inactive state, the dimerization loop locatedwithin domain 2 is not exposed and therefore unavailable fordimerization with other co-receptors (including, but not limited to,HER1, HER2 and HER4). In some embodiments, the antibody or fragmentthereof binds to human HER3 protein having a conformational epitopecomprising (i) HER3 amino acid residues 265-277 and 315 (of domain 2)and (ii) HER3 amino acid residues 571, 582-584, 596-597, 600-602,609-615 (of domain 4) of SEQ ID NO: 1, or a subset thereof. In someembodiments, the antibody or fragment thereof binds to amino acidswithin or overlapping amino acid residues 265-277 and 315 (of domain 2)and (ii) HER3 amino acid residues 571, 582-584, 596-597, 600-602,609-615 (of domain 4) of SEQ ID NO: 1. In some embodiments, the antibodyor fragment thereof binds to amino acids within (and/or amino acidsequences consisting of) amino acids 265-277 and 315 (of domain 2) and(ii) HER3 amino acid residues 571, 582-584, 596-597, 600-602, 609-615(of domain 4) of SEQ ID NO: 1, or a subset thereof. In some embodiments,the antibody or fragment thereof binds to the conformational epitopesuch that it restricts the mobility of domain 2 and domain 4,stabilizing it in an inactive or closed conformation. The failure toform the active conformation results in failure to activate signaltransduction. In some embodiments, the antibody or fragment thereofbinds to the conformational epitope such that it occludes thedimerization loop within domain 2, thereby rendering it unavailable forreceptor-receptor interaction. The failure to form homo- or heterodimersresults in failure to activate signal transduction.

In another aspect, the antibody or fragment thereof binds aconformational epitope of HER receptor, such as a HER3 receptor. In oneembodiment, the antibody or fragment thereof stabilizes the HER3receptor in the inactive state. In another embodiment, the antibody orfragment thereof binds to the active state of the HER3 receptor anddrives it into the inactive state as the inactive state. Thus, theantibody or fragment thereof can bind to either the active or inactivestate of HER3, but favors the formation of the inactive state and drivesthe active state of HER3 into the inactive state, resulting in a failureto activate signal transduction.

In another aspect, the antibody or fragment thereof binds aconformational epitope of HER receptor, such as a HER3 receptor wherebinding of the antibody or fragment thereof stabilizes the HER3 receptorin an inactive state such that the HER3 receptor fails to dimerize witha co-receptor to form a receptor-receptor complex. The failure to form areceptor-receptor complex prevents activation of both ligand-dependentand ligand-independent signal transduction.

In another aspect, the antibody or fragment thereof binds aconformational epitope of HER receptor such as a HER3 receptor, wherebinding of the antibody or fragment thereof to the HER3 receptor allowsdimerization with a co-receptor to form an inactive receptor-receptorcomplex. The formation of the inactive receptor-receptor complexprevents activation of ligand-independent signal transduction. Forexample, in ligand-independent signal transduction, HER3 may exists inan inactive state, however the overexpression of HER2 causes HER2-HER3complex formation, however these resulting complexes are inactive andprevent activation of ligand-independent signal transduction.

The depicted structure also allows one to identify specific core HER3amino acid residues for the interaction interface of an antibody orfragment thereof (e.g., MOR09823) with HER3. This was defined asresidues that are within 5 Å of the MOR09823 protein VH chain. The coreresidues are as follows: Asn266, Lys267, Leu268, Thr269, Gln271, Glu273,Pro274, Asn275, Pro276, His277, Asn315, Asp571, Pro583, His584, Ala596,Lys597 (residues of SEQ ID NO: 1).

The structures can also used to identify boundary HER3 amino acidresidues for the interaction interface with an antibody or fragmentthereof (e.g., MOR09823). These residues can be HER3 residues that were5-8 Å from the MOR09823 protein VH chain. The boundary residues are asfollows: Pro262, Va264, Tyr265, Phe270, Leu272, Thr278, Lys314, Gly316,Glu321, Asn566, Ser568, Gly569, Ser570, Thr572, Arg580, Asp581, Gly582,Gly595, Gly598, Ile600 (residues of SEQ ID NO: 1).

The depicted structure also allows one to identify specific core HER3amino acid residues for the interaction interface of an antibody orfragment thereof (e.g., MOR09823) with HER3. This was defined asresidues that are within 5 Å of the MOR09823 protein VL chain. The coreresidues are as follows: Tyr265, Lys267, Leu268, Phe270, Gly582, Pro583,Lys597, Ile600, Lys602, Glu609, Arg611, Pro612, Cys613, His614, Glu615(residues of SEQ ID NO: 1).

The structures were also used to identify boundary HER3 amino acidresidues for the interaction interface with an antibody or fragmentthereof (e.g., MOR09823). These residues were HER3 residues that were5-8 Å from the MOR09823 protein VL chain. The boundary residues are asfollows: Asn266, Thr269, Asp571, Arg580, Asp581, His584, Pro590, Ala596,Pro599, Tyr601, Tyr603, Asp605, Gln607, Cys610, Asn616, Cys617, Cys621,Gly623, Pro624 (residues of SEQ ID NO: 1).

As can be seen in Tables 11 and 12 (MOR09823) and Tables 13 and 14(MOR09825), respectively, the heavy chain is mainly involved in theantigen binding protein's binding to amino acid residues within domain 2of the epitope with fewer interactions with amino acid residues ofdomain 4, while the light chain is mainly involved with binding to aminoacid residues within domain 4 of the epitope with fewer interactionswith amino acid residues within domain 2.

As such, one of skill in the art, given the present teachings, canpredict which residues and areas of the antigen binding proteins can bevaried without unduly interfering with the antigen binding protein'sability to bind to HER3.

Core interaction interface amino acids were determined as being allamino acid residues with at least one atom less than or equal to 5 Åfrom the HER3 partner protein. 5 Å was chosen as the core region cutoffdistance to allow for atoms within a van der Waals radius plus apossible water-mediated hydrogen bond. Boundary interaction interfaceamino acids were determined as all amino acid residues with at least oneatom less than or equal to 8 Å from the HER3 partner protein but notincluded in the core interaction list.

In some embodiments, any antigen binding protein that binds to, covers,or prevents MOR09823 from interacting with any of the above residues canbe employed to bind to or neutralize HER3. In some embodiments, theantibodies or fragments thereof binds to or interacts with at least oneof the following HER3 residues (SEQ ID NO: 1): Asn266, Lys267, Leu268,Thr269, Gln271, Glu273, Pro274, Asn275, Pro276, His277, Asn315, Asp571,Pro583, His584, Ala596, Lys597. In some embodiments, the antibodies andfragments thereof binds to or interacts with at least one of thefollowing HER3 residues (SEQ ID NO: 1): Tyr265, Lys267, Leu268, Phe270,Gly582, Pro583, Lys597, Ile600, Lys602, Glu609, Arg611, Pro612, Cys613,His614, Glu615. In some embodiments, the antibodies or fragments thereofbinds to or interacts with at least one of the following HER3 residues(SEQ ID NO: 1): Asn266, Lys267, Leu268, Thr269, Gln271, Glu273, Pro274,Asn275, Pro276, His277, Asn315, Asp571, Pro583, His584, Ala596, Lys597,Tyr265, Lys267, Leu268, Phe270, Gly582, Pro583, Lys597, Ile600, Lys602,Glu609, Arg611, Pro612, Cys613, His614, Glu615. In some embodiments, theantibodies or fragments thereof binds to or interacts with a combinationof the following HER3 residues (SEQ ID NO: 1): Asn266, Lys267, Leu268,Thr269, Gln271, Glu273, Pro274, Asn275, Pro276, His277, Asn315, Asp571,Pro583, His584, Ala596, Lys597, Tyr265, Lys267, Leu268, Phe270, Gly582,Pro583, Lys597, Ile600, Lys602, Glu609, Arg611, Pro612, Cys613, His614,Glu615. In some embodiments, the antibodies or fragments thereof bindsto or interacts with all of the following HER3 residues (SEQ ID NO: 1):Asn266, Lys267, Leu268, Thr269, Gln271, Glu273, Pro274, Asn275, Pro276,His277, Asn315, Asp571, Pro583, His584, Ala596, Lys597, Tyr265, Lys267,Leu268, Phe270, Gly582, Pro583, Lys597, Ile600, Lys602, Glu609, Arg611,Pro612, Cys613, His614, Glu615. In some embodiments, the antibody orfragment thereof is within 5 angstroms of one or more of the aboveresidues. In some embodiments, the antibody or fragment thereof is 5 to8 angstroms from one or more of the above residues. In some embodiments,the antibody or fragment thereof interacts, blocks, or is within 8angstroms of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25,30, 35, 40, 45, or 50 of the above residues.

The availability of 3D structures for the HER3 and the complex ofHER3:MOR09823, for example, provides the framework to explore other HER3antibodies in more detail. The 3D structure of HER3 allows the epitopesfor monoclonal antibodies to be mapped and their mode of actioninferred, since some inhibit, some stimulate and others have no effecton cell growth. The conformational epitope for MOR09823 has been locatedto the domains 2 and 4 of HER3. The availability of the 3D structures ofthis receptor will facilitate the determination of the precise mechanismof action of these inhibitory agents and the design of new approaches tointerfering with HER3 receptor function. In one embodiment, theantibodies of the invention bind to the same conformational epitope asMOR09823.

In some embodiments, the conformational epitope bound by any of theantibodies listed in Table 1 is especially useful. In certainembodiments, a HER3 conformational epitope can be utilized to isolateantibodies of fragments thereof that bind to HER3. In certainembodiments, a HER3 conformational epitope can be utilized to generateantibodies or fragments thereof which bind to HER3. In certainembodiments, a HER3 conformational epitope can be utilized as animmunogen to generate antibodies of fragments thereof that bind to theHER3 conformational epitope. In certain embodiments, a HER3conformational epitope can be administered to an animal, and antibodiesthat bind to HER3 can subsequently be obtained from the animal.

In some embodiments, the domain(s)/region(s) containing residues thatare in contact with or are buried by an antibody can be identified bymutating specific residues in HER3 (e.g., a wild-type antigen) anddetermining whether antibody or fragment thereof can bind the mutated orvariant HER3 protein or measure changes of affinity from wild-type. Bymaking a number of individual mutations, residues that play a directrole in binding or that are in sufficiently close proximity to theantibody such that a mutation can affect binding between the antibodyand antigen can be identified. From a knowledge of these amino acids,the domain(s) or region(s) of the antigen (HER3) that contain residuesin contact with the antibody or covered by the antibody can beelucidated. Mutagenesis using known techniques such as alanine-scanningcan help define functionally relevant epitopes. Mutagenesis utilizing anarginine/glutamic acid scanning protocol can also be employed (see,e.g., Nanevicz et al., (1995), J. Biol. Chem. 270(37):21619-21625 andZupnick et al., (2006), J. Biol. Chem. 281(29):20464-20473). In general,arginine and glutamic acids are substituted (typically individually) foran amino acid in the wild-type polypeptide because these amino acids arecharged and bulky and thus have the potential to disrupt binding betweenan antigen binding protein and an antigen in the region of the antigenwhere the mutation is introduced. Arginines that exist in the wild-typeantigen are replaced with glutamic acid. A variety of such individualmutants can be obtained and the collected binding results analyzed todetermine what residues affect binding. A series of mutant HER3 antigenscan be created, with each mutant antigen having a single mutation.Binding of each mutant HER3 antigen with various HER3 antibodies orfragments thereof can be measured and compared to the ability of theselected an antibody or fragments thereof to bind wild-type HER3 (SEQ IDNO: 1).

An alteration (for example a reduction or increase) in binding betweenan antibody or fragment thereof and a mutant or variant HER3 as usedherein means that there is a change in binding affinity (e.g., asmeasured by known methods such as Biacore testing or the bead basedassay described below in the examples), EC₅₀, and/or a change (forexample a reduction) in the total binding capacity of the antigenbinding protein (for example, as evidenced by a decrease in B_(max) in aplot of antigen binding protein concentration versus antigenconcentration). A significant alteration in binding indicates that themutated residue is involved in binding to the antibody or fragmentthereof.

In some embodiments, a significant reduction in binding means that thebinding affinity, EC₅₀, and/or capacity between an antibody or fragmentsthereof and a mutant HER3 antigen is reduced by greater than 10%,greater than 20%, greater than 40%, greater than 50%, greater than 55%,greater than 60%, greater than 65%, greater than 70%, greater than 75%,greater than 80%, greater than 85%, greater than 90% or greater than 95%relative to binding between the an antibody or fragment thereof and awild type HER3 (e.g., SEQ ID NO: 1).

In some embodiments, binding of an antibody or fragments thereof issignificantly reduced or increased for a mutant HER3 protein having oneor more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) mutations ascompared to a wild-type HER3 protein (e.g., SEQ ID NO: 1).

Although the variant forms are referenced with respect to the wild-typesequence shown in SEQ ID NO: 1, it will be appreciated that in anallelic or splice variants of HER3 the amino acids could differ.Antibodies or fragments thereof showing significantly altered binding(e.g., lower or higher binding) for such allelic forms of HER3 are alsocontemplated.

In addition to the general structural aspects of antibodies, the morespecific interaction between the paratope and the epitope may beexamined through structural approaches. In one embodiment, the structureof the CDRs contribute to a paratope, through which an antibody is ableto bind to an epitope. The shape of such a paratope may be determined ina number of ways. Traditional structural examination approaches can beused, such as NMR or x-ray crystallography. These approaches can examinethe shape of the paratope alone, or while it is bound to the epitope.Alternatively, molecular models may be generated in silico. A structurecan be generated through homology modeling, aided with a commercialpackage, such as InsightII modeling package from Accelrys (San Diego,Calif.). Briefly, one can use the sequence of the antibody to beexamined to search against a database of proteins of known structures,such as the Protein Data Bank. After one identifies homologous proteinswith known structures, these homologous proteins are used as modelingtemplates. Each of the possible templates can be aligned, thus producingstructure based sequence alignments among the templates. The sequence ofthe antibody with the unknown structure can then be aligned with thesetemplates to generate a molecular model for the antibody with theunknown structure. As will be appreciated by one of skill in the art,there are many alternative methods for generating such structures insilico, any of which may be used. For instance, a process similar to theone described in Hardman et al., issued U.S. Pat. No. 5,958,708employing QUANTA (Polygen Corp., Waltham, Mass.) and CHARM (Brooks etal., (1983), J. Comp. Chem. 4:187) may be used (hereby incorporated inits entirety by reference).

Not only is the shape of the paratope important in determining whetherand how well a possible paratope will bind to an epitope, but theinteraction itself, between the epitope and the paratope is a source ofgreat information in the design of variant antibodies. As appreciated byone of skill in the art, there are a variety of ways in which thisinteraction can be studied. One way is to use the structural modelgenerated, perhaps as described above, and then to use a program such asInsightII (Accelrys, San Diego, Calif.), which has a docking module,which, among other things, is capable of performing a Monte Carlo searchon the conformational and orientational spaces between the paratope andits epitope. The result is that one is able to estimate where and howthe epitope interacts with the paratope. In one embodiment, only afragment, or variant, of the epitope is used to assist in determiningthe relevant interactions. In one embodiment, the entire epitope is usedin the modeling of the interaction between the paratope and the epitope.

Through the use of these modelled structures, one is able to predictwhich residues are the most important in the interaction between theepitope and the paratope. Thus, in one embodiment, one is able toreadily select which residues to change in order to alter the bindingcharacteristics of the antibody. For instance, it may be apparent fromthe docking models that the side chains of certain residues in theparatope may sterically hinder the binding of the epitope, thus alteringthese residues to residues with smaller side chains may be beneficial.One can determine this in many ways. For example, one may simply look atthe two models and estimate interactions based on functional groups andproximity. Alternatively, one may perform repeated pairings of epitopeand paratope, as described above, in order to obtain more favorableenergy interactions. One can also determine these interactions for avariety of variants of the antibody to determine alternative ways inwhich the antibody may bind to the epitope. One can also combine thevarious models to determine how one should alter the structure of theantibodies in order to obtain an antibody with the particularcharacteristics that are desired.

The models determined above can be tested through various techniques.For example, the interaction energy can determined with the programsdiscussed above in order to determine which of the variants to furtherexamine. Also, coulumbic and van der Waals interactions are used todetermine the interaction energies of the epitope and the variantparatopes. Also site directed mutagenesis is used to see if predictedchanges in antibody structure actually result in the desired changes inbinding characteristics. Alternatively, changes may be made to theepitope to verify that the models are correct or to determine generalbinding themes that may be occurring between the paratope and theepitope.

As will be appreciated by one of skill in the art, while these modelswill provide the guidance necessary to make the antibodies and variantsthereof of the present embodiments, it may still be desirable to performroutine testing of the in silico models, perhaps through in vitrostudies. In addition, as will be apparent to one of skill in the art,any modification may also have additional side effects on the activityof the antibody. For instance, while any alteration predicted to resultin greater binding, may induce greater binding, it may also cause otherstructural changes which might reduce or alter the activity of theantibody. The determination of whether or not this is the case isroutine in the art and can be achieved in many ways. For example, theactivity can be tested through an ELISA test. Alternatively, the samplescan be tested through the use of a surface plasmon resonance device.

HER3 Antibodies

The present invention provides antibodies that recognize aconformational epitope of HER3. The invention is based on the surprisingfinding that a class of antibodies against HER3, block bothligand-dependent and ligand-independent HER3 signal transductionpathways. A class of antibodies that bind to the particular conformationepitope of HER3 is disclosed in Table 1. In one embodiment, theantibodies inhibit both ligand-dependent and ligand-independent HER3signalling. In another embodiment, the antibodies bind to HER3 and donot block HER ligand binding to the ligand binding site (i.e. bothligand and antibody can bind HER3 concurrently).

The present invention provides antibodies that specifically bind a HER3protein (e.g., human and/or cynomologus HER3), said antibodiescomprising a VH domain having an amino acid sequence of SEQ ID NO: 15,33, 51, 69, 87, 105, 123, 141, 159, 177, 195, 213, 231, 249, 267, 285,303, 321, 339, 357, and 375. The present invention provides antibodiesthat specifically bind a HER3 protein (e.g., human and/or cynomologusHER3), said antibodies comprising a VL domain having an amino acidsequence of SEQ ID NO: 14, 32, 50, 68, 86, 104, 122, 140, 158, 176, 194,212, 230, 248, 266, 284, 302, 320, 338, 356, and 374. The presentinvention also provides antibodies that specifically bind to a HER3protein (e.g., human and/or cynomologus HER3), said antibodiescomprising a VH CDR having an amino acid sequence of any one of the VHCDRs listed in Table 1, infra. In particular, the invention providesantibodies that specifically bind to a HER3 protein (e.g., human and/orcynomologus HER3), said antibodies comprising (or alternatively,consisting of) one, two, three, four, five or more VH CDRs having anamino acid sequence of any of the VH CDRs listed in Table 1, infra.Other antibodies of the invention include amino acids that have beenmutated, yet have at least 60, 70, 80, 90, 95, or 98 percent identity inthe CDR regions with the CDR regions depicted in the sequences describedin Table 1. In some embodiments, it includes mutant amino acid sequenceswherein no more than 1, 2, 3, 4 or 5 amino acids have been mutated inthe CDR regions when compared with the CDR regions depicted in thesequence described Table 1, while still maintaining their specificityfor the original antibody's epitope

Other antibodies of the invention include amino acids that have beenmutated, yet have at least 60, 70, 80, 90, 95, or 98 percent identity inthe framework regions with the framework regions depicted in thesequences described in Table 1. In some embodiments, it includes mutantamino acid sequences wherein no more than 1, 2, 3, 4, 5, 6, or 7 aminoacids have been mutated in the framework regions when compared with theframework regions depicted in the sequence described Table 1, whilestill maintaining their specificity for the original antibody's epitope.The present invention also provides nucleic acid sequences that encodeVH, VL, the full length heavy chain, and the full length light chain ofthe antibodies that specifically bind to a HER3 protein (e.g., humanand/or cynomologus HER3).

The HER3 antibodies of the invention bind to the conformational epitopeof HER3 comprising amino acid residues from domain 2 and domain 4 ofHER3.

TABLE 1 Examples of HER3 Antibodies of the Present InventionSEQ ID NUMBER Ab region MOR09823 SEQ ID NO: 2 (Kabat) HCDR1 SYAMSSEQ ID NO: 3 (Kabat) HCDR2 VTGAVGRTYYPDSVKG SEQ ID NO: 4 (Kabat) HCDR3WGDEGFDI SEQ ID NO: 5 (Kabat) LCDR1 RASQGISNWLA SEQ ID NO: 6 (Kabat)LCDR2 GASSLQS SEQ ID NO: 7 (Kabat) LCDR3 QQYSSFPTTSEQ ID NO: 8 (Chothia) HCDR1 GFTFSSY SEQ ID NO: 9 (Chothia) HCDR2 GAVGRSEQ ID NO: 10 (Chothia) HCDR3 WGDEGFDI SEQ ID NO: 11 (Chothia) LCDR1SQGISNW SEQ ID NO: 12 (Chothia) LCDR2 GAS SEQ ID NO: (Chothia) 13 LCDR3YSSFPT SEQ ID NO: 14 VLDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQYSSFPTTFGQ GTKVEIK SEQ ID NO: 15VH QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVTGAVGRTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGD EGFDIWGQGTLVTVSSSEQ ID NO: 16 DNA VLGATATCCAGATGACCCAGAGCCCGTCTAGCCTGAGCGCGAGCGTGGGTGATCGTGTGACCATTACCTGCAGAGCGAGCCAGGGTATTTCTAATTGGCTGGCTTGGTACCAGCAGAAACCAGGTAAAGCACCGAAACTATTAATTTATGGTGCTTCTTCTTTGCAAAGCGGGGTCCCGTCCCGTTTTAGCGGCTCTGGATCCGGCACTGATTTTACCCTGACCATTAGCAGCCTGCAACCTGAAGACTTTGCGGTTTATTATTGCCAGCAGTATTCTTCTTTTCCTACTACCTTTGGCCAGGGTACGAAAGTTGAAATTAAA SEQ ID NO: 17 DNA VHCAGGTGCAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCCTGCGTCTGAGCTGCGCGGCCTCCGGATTTACCTTTAGCAGCTATGCGATGAGCTGGGTGCGCCAAGCCCCTGGGAAGGGTCTCGAGTGGGTGAGCGTTACTGGTGCTGTTGGTCGTACTTATTATCCTGATTCTGTTAAGGGTCGTTTTACCATTTCACGTGATAATTCGAAAAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGCGCGTTGGGGTGATGAGGGTTTTGATATTTGGGGCCAAGGCACCCTGGTGACGGTTAGCTCA SEQ ID NO: 18 Light KappaDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQYSSFPTTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECSEQ ID NO: 19 Heavy IgG1QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVTGAVGRTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPMDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK MOR09824SEQ ID NO: 20 (Kabat) HCDR1 SYAMS SEQ ID NO: 21 (Kabat) HCDR2VISAWGHVKYYADSVKG SEQ ID NO: 22 (Kabat) HCDR3 WGDEGFDISEQ ID NO: 23 (Kabat) LCDR1 RASQGISNWLA SEQ ID NO: 24 (Kabat) LCDR2GASSLQS SEQ ID NO: 25 (Kabat) LCDR3 QQYSSFPTT SEQ ID NO: 26 (Chothia)HCDR1 GFTFSSY SEQ ID NO: 27 (Chothia) HCDR2 SAWGHVSEQ ID NO: 28 (Chothia) HCDR3 WGDEGFDI SEQ ID NO: 29 (Chothia) LCDR1SQGISNW SEQ ID NO: 30 (Chothia) LCDR2 GAS SEQ ID NO: 31 (Chothia) LCDR3YSSFPT SEQ ID NO: 32 VLDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQYSSFPTTFGQ GTKVEIK SEQ ID NO: 33VH QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVISAWGHVKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWG DEGFDIWGQGTLVTVSSSEQ ID NO: 34 DNA VLGATATCCAGATGACCCAGAGCCCGTCTAGCCTGAGCGCGAGCGTGGGTGATCGTGTGACCATTACCTGCAGAGCGAGCCAGGGTATTTCTAATTGGCTGGCTTGGTACCAGCAGAAACCAGGTAAAGCACCGAAACTATTAATTTATGGTGCTTCTTCTTTGCAAAGCGGGGTCCCGTCCCGTTTTAGCGGCTCTGGATCCGGCACTGATTTTACCCTGACCATTAGCAGCCTGCAACCTGAAGACTTTGCGGTTTATTATTGCCAGCAGTATTCTTCTTTTCCTACTACCTTTGGCCAGGGTACGAAAGTTGAAATTAAA SEQ ID NO: 35 DNA VHCAGGTGCAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCCTGCGTCTGAGCTGCGCGGCCTCCGGATTTACCTTTAGCAGCTATGCGATGAGCTGGGTGCGCCAAGCCCCTGGGAAGGGTCTCGAGTGGGTGAGCGTTATTTCTGCTTGGGGTCATGTTAAGTATTATGCTGATTCTGTTAAGGGTCGTTTTACCATTTCACGTGATAATTCGAAAAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGCGCGTTGGGGTGATGAGGGTTTTGATATTTGGGGCCAAGGCACCCTGGTGACGGTTAGCTCA SEQ ID NO: 36 Light KappaDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQYSSFPTTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECSEQ ID NO: 37 Heavy IgG1QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVISAWGHVKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK MOR09825SEQ ID NO: 38 (Kabat) HCDR1 SYAMS SEQ ID NO: 39 (Kabat) HCDR2AINSQGKSTYYADSVKG SEQ ID NO: 40 (Kabat) HCDR3 WGDEGFDISEQ ID NO: 41 (Kabat) LCDR1 RASQGISNWLA SEQ ID NO: 42 (Kabat) LCDR2GASSLQS SEQ ID NO: 43 (Kabat) LCDR3 QQYSSFPTT SEQ ID NO: 44 (Chothia)HCDR1 GFTFSSY SEQ ID NO: 45 (Chothia) HCDR2 NSQGKSSEQ ID NO: 46 (Chothia) HCDR3 WGDEGFDI SEQ ID NO: 47 (Chothia) LCDR1SQGISNW SEQ ID NO: 48 (Chothia) LCDR2 GAS SEQ ID NO: 49 (Chothia) LCDR3YSSFPT SEQ ID NO: 50 VLDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQYSSFPTTFGQ GTKVEIK SEQ ID NO: 51VH QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINSQGKSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWG DEGFDIWGQGTLVTVSSSEQ ID NO: 52 DNA VLGATATCCAGATGACCCAGAGCCCGTCTAGCCTGAGCGCGAGCGTGGGTGATCGTGTGACCATTACCTGCAGAGCGAGCCAGGGTATTTCTAATTGGCTGGCTTGGTACCAGCAGAAACCAGGTAAAGCACCGAAACTATTAATTTATGGTGCTTCTTCTTTGCAAAGCGGGGTCCCGTCCCGTTTTAGCGGCTCTGGATCCGGCACTGATTTTACCCTGACCATTAGCAGCCTGCAACCTGAAGACTTTGCGGTTTATTATTGCCAGCAGTATTCTTCTTTTCCTACTACCTTTGGCCAGGGTACGAAAGTTGAAATTAAA SEQ ID NO: 53 DNA VHCAGGTGCAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCCTGCGTCTGAGCTGCGCGGCCTCCGGATTTACCTTTAGCAGCTATGCGATGAGCTGGGTGCGCCAAGCCCCTGGGAAGGGTCTCGAGTGGGTGAGCGCTATTAATTCTCAGGGTAAGTCTACTTATTATGCTGATTCTGTTAAGGGTCGTTTTACCATTTCACGTGATAATTCGAAAAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGCGCGTTGGGGTGATGAGGGTTTTGATATTTGGGGCCAAGGCACCCTGGTGACGGTTAGCTCA SEQ ID NO: 54 Light KappaDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQYSSFPTTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECSEQ ID NO: 55 Heavy IgG1QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINSQGKSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK MOR09974SEQ ID NO: 56 (Kabat) HCDR1 SYAMS SEQ ID NO: 57 (Kabat) HCDR2VINPSGNFTNYADSVKG SEQ ID NO: 58 (Kabat) HCDR3 WGDEGFDISEQ ID NO: 59 (Kabat) LCDR1 RASQGISNWLA SEQ ID NO: 60 (Kabat) LCDR2GASSLQS SEQ ID NO: 61 (Kabat) LCDR3 QQYSSFPTT SEQ ID NO: 62 (Chothia)HCDR1 GFTFSSY SEQ ID NO: 63 (Chothia) HCDR2 NPSGNFSEQ ID NO: 64 (Chothia) HCDR3 WGDEGFDI SEQ ID NO: 65 (Chothia) LCDR1SQGISNW SEQ ID NO: 66 (Chothia) LCDR2 GAS SEQ ID NO: 67 (Chothia) LCDR3YSSFPT SEQ ID NO: 68 VLDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQYSSFPTTFGQ GTKVEIK SEQ ID NO: 69VH QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVINPSGNFTNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWG DEGFDIWGQGTLVTVSSSEQ ID NO: 70 DNA VLGATATCCAGATGACCCAGAGCCCGTCTAGCCTGAGCGCGAGCGTGGGTGATCGTGTGACCATTACCTGCAGAGCGAGCCAGGGTATTTCTAATTGGCTGGCTTGGTACCAGCAGAAACCAGGTAAAGCACCGAAACTATTAATTTATGGTGCTTCTTCTTTGCAAAGCGGGGTCCCGTCCCGTTTTAGCGGCTCTGGATCCGGCACTGATTTTACCCTGACCATTAGCAGCCTGCAACCTGAAGACTTTGCGGTTTATTATTGCCAGCAGTATTCTTCTTTTCCTACTACCTTTGGCCAGGGTACGAAAGTTGAAATTAAA SEQ ID NO: 71 DNA VHCAGGTGCAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCCTGCGTCTGAGCTGCGCGGCCTCCGGATTTACCTTTAGCAGCTATGCGATGAGCTGGGTGCGCCAAGCCCCTGGGAAGGGTCTCGAGTGGGTGAGCGTTATTAATCCTTCTGGTAATTTTACTAATTATGCTGATTCTGTTAAGGGTCGTTTTACCATTTCACGTGATAATTCGAAAAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGCGCGTTGGGGTGATGAGGGTTTTGATATTTGGGGCCAAGGCACCCTGGTGACGGTTAGCTCA SEQ ID NO: 72 Light KappaDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQYSSFPTTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECSEQ ID NO: 73 Heavy IgG1QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVINPSGNFTNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK MOR10452SEQ ID NO: 74 (Kabat) HCDR1 SYAMS SEQ ID NO: 75 (Kabat) HCDR2NTSPIGYTYYAGSVKG SEQ ID NO: 76 (Kabat) HCDR3 WGDEGFDISEQ ID NO: 77 (Kabat) LCDR1 RASQGISNWLA SEQ ID NO: 78 (Kabat) LCDR2GASSLQS SEQ ID NO: 79 (Kabat) LCDR3 QQYSSFPTT SEQ ID NO: 80 (Chothia)HCDR1 GFTFSSY SEQ ID NO: 81 (Chothia) HCDR2 SPIGYSEQ ID NO: 82 (Chothia) HCDR3 WGDEGFDI SEQ ID NO: 83 (Chothia) LCDR1SQGISNW SEQ ID NO: 84 (Chothia) LCDR2 GAS SEQ ID NO: 85 (Chothia) LCDR3YSSFPT SEQ ID NO: 86 VLDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQYSSFPTTFGQ GTKVEIK SEQ ID NO: 87VH QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNTSPIGYTYYAGSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGD EGFDIWGQGTLVTVSSSEQ ID NO: 88 DNA VLGATATCCAGATGACCCAGAGCCCGTCTAGCCTGAGCGCGAGCGTGGGTGATCGTGTGACCATTACCTGCAGAGCGAGCCAGGGTATTTCTAATTGGCTGGCTTGGTACCAGCAGAAACCAGGTAAAGCACCGAAACTATTAATTTATGGTGCTTCTTCTTTGCAAAGCGGGGTCCCGTCCCGTTTTAGCGGCTCTGGATCCGGCACTGATTTTACCCTGACCATTAGCAGCCTGCAACCTGAAGACTTTGCGGTTTATTATTGCCAGCAGTATTCTTCTTTTCCTACTACCTTTGGCCAGGGTACGAAAGTTGAAATTAAA SEQ ID NO: 89 DNA VHCAGGTGCAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCCTGCGTCTGAGCTGCGCGGCCTCCGGATTTACCTTTAGCAGCTATGCGATGAGCTGGGTGCGCCAAGCCCCTGGGAAGGGTCTCGAGTGGGTGAGCAATACTTCTCCTATTGGTTATACTTATTATGCTGGTTCTGTTAAGGGTCGTTTTACCATTTCACGTGATAATTCGAAAAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGCGCGTTGGGGTGATGAGGGTTTTGATATTTGGGGCCAAGGCACCCTGGTGACGGTTAGCTCA SEQ ID NO: 90 Light KappaDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQYSSFPTTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEASEQ ID NO: 91 Heavy ChainQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSN (only VH andTSPIGYTYYAGSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGD CH1 domains)EGFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSMOR10701 SEQ ID NO: 92 (Kabat) HCDR1 SYAMS SEQ ID NO: 93 (Kabat) HCDR2VTGAVGRSTYYPDSVKG SEQ ID NO: 94 (Kabat) HCDR3 WGDEGFDISEQ ID NO: 95 (Kabat) LCDR1 RASQGISNWLA SEQ ID NO: 96 (Kabat) LCDR2GASSLQS SEQ ID NO: 97 (Kabat) LCDR3 QQYSSFPTT SEQ ID NO: 98 (Chothia)HCDR1 GFTFSSY SEQ ID NO: 99 (Chothia) HCDR2 GAVGRSSEQ ID NO: 100 (Chothia) HCDR3 WGDEGFDI SEQ ID NO: 101 (Chothia) LCDR1SQGISNW SEQ ID NO: 102 (Chothia) LCDR2 GAS SEQ ID NO: 103 (Chothia)LCDR3 YSSFPT SEQ ID NO: 104 VLDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTTFGQ GTKVEIKSEQ ID NO: 105 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVTGAVGRSTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWG DEGFDIWGQGTLVTVSSSEQ ID NO: 106 DNA VLGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGAGTGACCATCACCTGTCGGGCCAGCCAGGGCATCAGCAACTGGCTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGGCGCCAGCTCCCTGCAGAGCGGCGTGCCAAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAGCAGCTTCCCCACCACCTTCGGCCAGGGCACCAAGGTGGAAATCAAG SEQ ID NO: 107 DNA VHGAGGTGCAATTGCTGGAAAGCGGCGGAGGCCTGGTGCAGCCTGGCGGCAGCCTGAGACTGTCTTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTCCGCCAGGCCCCTGGCAAGGGACTGGAATGGGTGTCCGTGACAGGCGCCGTGGGCAGAAGCACCTACTACCCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTGCCAGATGGGGCGACGAGGGCTTCGACATCTGGGGCCAGGGCACCCTGGTCACCGTCAGCTCA SEQ ID NO: 108 Light KappaDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKESTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECSEQ ID NO: 109 Heavy IgG1EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVTGAVGRSTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK MOR10702SEQ ID NO: 110 (Kabat) HCDR1 SYAMS SEQ ID NO: 111 (Kabat) HCDR2VISAWGHVKYYADSVKG SEQ ID NO: 112 (Kabat) HCDR3 WGDEGFDISEQ ID NO: 113 (Kahat) LCDR1 RASQGISNWLA SEQ ID NO: 114 (Kabat) LCDR2GASSLQS SEQ ID NO: 115 (Kabat) LCDR3 QQYSSFPTT SEQ ID NO: 116 (Chothia)HCDR1 GFTFSSY SEQ ID NO: 117 (Chothia) HCDR2 SAWGHVSEQ ID NO: 118 (Chothia) HCDR3 WGDEGFDI SEQ ID NO: 119 (Chothia) LCDR1SQGISNW SEQ ID NO: 120 (Chothia) LCDR2 GAS SEQ ID NO: 121 (Chothia)LCDR3 YSSFPT SEQ ID NO: 122 VLDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTTFGQ GTKVEIKSEQ ID NO: 123 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVISAWGHVKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWG DEGFDIWGQGTLVTVSSSEQ ID NO: 124 DNA VLGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGAGTGACCATCACCTGTCGGGCCAGCCAGGGCATCAGCAACTGGCTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGGCGCCAGCTCCCTGCAGAGCGGCGTGCCAAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAGCAGCTTCCCCACCACCTTCGGCCAGGGCACCAAGGTGGAAATCAAG SEQ ID NO: 125 DNA VHGAGGTGCAATTGCTGGAAAGCGGCGGAGGCCTGGTGCAGCCTGGCGGCAGCCTGAGACTGTCTTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTCCGCCAGGCCCCTGGCAAGGGACTGGAATGGGTGTCCGTGATCAGCGCCTGGGGCCACGTGAAGTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTGCCAGATGGGGCGACGAGGGCTTCGACATCTGGGGCCAGGGCACCCTGGTCACCGTCAGCTCA SEQ ID NO: 126 Light KappaDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECSEQ ID NO: 127 Heavy IgG1EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVISAWGHVKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK MOR10703SEQ ID NO: 128 (Kabat) HCDR1 SYAMS SEQ ID NO: 129 (Kabat) HCDR2AINSQGKSTYYADSVKG SEQ ID NO: 130 (Kabat) HCDR3 WGDEGFDISEQ ID NO: 131 (Kabat) LCDR1 RASQGISNWLA SEQ ID NO: 132 (Kabat) LCDR2GASSLQS SEQ ID NO: 133 (Kabat) LCDR3 QQYSSFPTT SEQ ID NO: 134 (Chothia)HCDR1 GFTFSSY SEQ ID NO: 135 (Chothia) HCDR2 NSQGKSSEQ ID NO: 136 (Chothia) HCDR3 WGDEGFDI SEQ ID NO: 137 (Chothia) LCDR1SQGISNW SEQ ID NO: 138 (Chothia) LCDR2 GAS SEQ ID NO: 139 (Chothia)LCDR3 YSSFPT SEQ ID NO: 140 VLDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTTFGQ GTKVEIKSEQ ID NO: 141 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINSQGKSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWG DEGFDIWGQGTLVTVSSSEQ ID NO: 142 DNA VLGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGAGTGACCATCACCTGTCGGGCCAGCCAGGGCATCAGCAACTGGCTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGGCGCCAGCTCCCTGCAGAGCGGCGTGCCAAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAGCAGCTTCCCCACCACCTTCGGCCAGGGCACCAAGGTGGAAATCAAG SEQ ID NO: 143 DNA VHGAGGTGCAATTGCTGGAAAGCGGCGGAGGCCTGGTGCAGCCTGGCGGCAGCCTGAGACTGTCTTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTCCGCCAGGCCCCTGGCAAGGGACTGGAATGGGTGTCCGCCATCAACAGCCAGGGCAAGAGCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTGCCAGATGGGGCGACGAGGGCTTCGACATCTGGGGCCAGGGCACCCTGGTCACCGTCAGCTCA SEQ ID NO: 144 Light KappaDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECSEQ ID NO: 145 Heavy IgG1EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINSQGKSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK MOR10703 N52SSEQ ID NO: 146 (Kabat) HCDR1 SYAMS SEQ ID NO: 147 (Kabat) HCDR2 AI SSQGKSTYYADSVKG SEQ ID NO: 148 (Kabat) HCDR3 WGDEGFDISEQ ID NO: 149 (Kabat) LCDR1 RASQGISNWLA SEQ ID NO: 150 (Kabat) LCDR2GASSLQS SEQ ID NO: 151 (Kabat) LCDR3 QQYSSFPTT SEQ ID NO: 152 (Chothia)HCDR1 GFTFSSY SEQ ID NO: 153 (Chothia) HCDR2 S SQGKSSEQ ID NO: 154 (Chothia) HCDR3 WGDEGFDI SEQ ID NO: 155 (Chothia) LCDR1SQGISNW SEQ ID NO: 156 (Chothia) LCDR2 GAS SEQ ID NO: 157 (Chothia)LCDR3 YSSFPT SEQ ID NO: 158 VLDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTTFGQGTKVEIK SEQ ID NO: 159 VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAI S SQGKSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDIWGQG TLVTVSSSEQ ID NO: 160 DNA VLGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGAGTGACCATCACCTGTCGGGCCAGCCAGGGCATCAGCAACTGGCTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGGCGCCAGCTCCCTGCAGAGCGGCGTGCCAAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAGCAGCTTCCCCACCACCTTCGGCCAGGGCACCAAGGTGGAAATCAAG SEQ ID NO: 161 DNA VHGAGGTGCAATTGCTGGAAAGCGGCGGAGGCCTGGTGCAGCCTGGCGGCAGCCTGAGACTGTCTTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTCCGCCAGGCCCCTGGCAAGGGACTGGAATGGGTGTCCGCCATCAGCAGCCAGGGCAAGAGCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTGCCAGATGGGGCGACGAGGGCTTCGACATCTGGGGCCAGGGCACCCTGGTCACCGTCAGCTCA SEQ ID NO: 162 Light KappaDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 163Heavy IgG1 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAI S SQGKSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGKMOR10703 N52G SEQ ID NO: 164 (Kabat) HCDR1 SYAMS SEQ ID NO: 165 (Kabat)HCDR2 AI G SQGKSTYYADSVKG SEQ ID NO: 166 (Kabat) HCDR3 WGDEGFDISEQ ID NO: 167 (Kabat) LCDR1 RASQGISNWLA SEQ ID NO: 168 (Kabat) LCDR2GASSLQS SEQ ID NO: 169 (Kabat) LCDR3 QQYSSFPTT SEQ ID NO: 170 (Chothia)HCDR1 GFTFSSY SEQ ID NO: 171 (Chothia) HCDR2 G SQGKSSEQ ID NO: 172 (Chothia) HCDR3 WGDEGFDI SEQ ID NO: 173 (Chothia) LCDR1SQGISNW SEQ ID NO: 174 (Chothia) LCDR2 GAS SEQ ID NO: 175 (Chothia)LCDR3 YSSFPT SEQ ID NO: 176 VLDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTTFGQGTKVEIK SEQ ID NO: 177 VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAI G SQGKSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDIWGQG TLVTVSSSEQ ID NO: 178 DNA VLGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGAGTGACCATCACCTGTCGGGCCAGCCAGGGCATCAGCAACTGGCTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGGCGCCAGCTCCCTGCAGAGCGGCGTGCCAAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAGCAGCTTCCCCACCACCTTCGGCCAGGGCACCAAGGTGGAAATCAAG SEQ ID NO: 179 DNA VHGAGGTGCAATTGCTGGAAAGCGGCGGAGGCCTGGTGCAGCCTGGCGGCAGCCTGAGACTGTCTTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTCCGCCAGGCCCCTGGCAAGGGACTGGAATGGGTGTCCGCCATCGGCAGCCAGGGCAAGAGCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTGCCAGATGGGGCGACGAGGGCTTCGACATCTGGGGCCAGGGCACCCTGGTCACCGTCAGCTCA SEQ ID NO: 180 Light KappaDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 181Heavy IgG1 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAI G SQGKSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGKMOR10703 N52S_S52aN SEQ ID NO: 182 (Kabat) HCDR1 SYAMSSEQ ID NO: 183 (Kabat) HCDR2 AI SN QGKSTYYADSVKG SEQ ID NO: 184 (Kabat)HCDR3 WGDEGFDI SEQ ID NO: 185 (Kabat) LCDR1 RASQGISNWLASEQ ID NO: 186 (Kabat) LCDR2 GASSLQS SEQ ID NO: 187 (Kabat) LCDR3QQYSSFPTT SEQ ID NO: 188 (Chothia) HCDR1 GFTFSSYSEQ ID NO: 189 (Chothia) HCDR2 SN QGKS SEQ ID NO: 190 (Chothia) HCDR3WGDEGFDI SEQ ID NO: 191 (Chothia) LCDR1 SQGISNW SEQ ID NO: 192 (Chothia)LCDR2 GAS SEQ ID NO: 193 (Chothia) LCDR3 YSSFPT SEQ ID NO: 194 VLDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTTFGQGTKVEIK SEQ ID NO: 195 VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAI SN QGKSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDIWGQG TLVTVSSSEQ ID NO: 196 DNA VLGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGAGTGACCATCACCTGTCGGGCCAGCCAGGGCATCAGCAACTGGCTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGGCGCCAGCTCCCTGCAGAGCGGCGTGCCAAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAGCAGCTTCCCCACCACCTTCGGCCAGGGCACCAAGGTGGAAATCAAG SEQ ID NO: 197 DNA VHGAGGTGCAATTGCTGGAAAGCGGCGGAGGCCTGGTGCAGCCTGGCGGCAGCCTGAGACTGTCTTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTCCGCCAGGCCCCTGGCAAGGGACTGGAATGGGTGTCCGCCATCAGCAACCAGGGCAAGAGCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTGCCAGATGGGGCGACGAGGGCTTCGACATCTGGGGCCAGGGCACCCTGGTCACCGTCAGCTCA SEQ ID NO: 198 Light KappaDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 199Heavy IgG1 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAI SN QGKSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGKMOR10703 A50V_N52S SEQ ID NO: 200 (Kabat) HCDR1 SYAMSSEQ ID NO: 201 (Kabat) HCDR2 V I S SQGKSTYYADSVKG SEQ ID NO: 202 (Kabat)HCDR3 WGDEGFDI SEQ ID NO: 203 (Kabat) LCDR1 RASQGISNWLASEQ ID NO: 204 (Kabat) LCDR2 GASSLQS SEQ ID NO: 205 (Kabat) LCDR3QQYSSFPTT SEQ ID NO: 206 (Chothia) HCDR1 GFTFSSYSEQ ID NO: 207 (Chothia) HCDR2 S SQGKS SEQ ID NO: 208 (Chothia) HCDR3WGDEGFDI SEQ ID NO: 209 (Chothia) LCDR1 SQGISNW SEQ ID NO: 210 (Chothia)LCDR2 GAS SEQ ID NO: 211 (Chothia) LCDR3 YSSFPT SEQ ID NO: 212 VLDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTTFGQGTKVEIK SEQ ID NO: 213 VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVS V I S SQGKSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDIWGQG TLVTVSSSEQ ID NO: 214 DNA VLGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGAGTGACCATCACCTGTCGGGCCAGCCAGGGCATCAGCAACTGGCTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGGCGCCAGCTCCCTGCAGAGCGGCGTGCCAAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAGCAGCTTCCCCACCACCTTCGGCCAGGGCACCAAGGTGGAAATCAAG SEQ ID NO: 215 DNA VHGAGGTGCAATTGCTGGAAAGCGGCGGAGGCCTGGTGCAGCCTGGCGGCAGCCTGAGACTGTCTTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTCCGCCAGGCCCCTGGCAAGGGACTGGAATGGGTGTCCGTCATCAGCAGCCAGGGCAAGAGCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTGCCAGATGGGGCGACGAGGGCTTCGACATCTGGGGCCAGGGCACCCTGGTCACCGTCAGCTCA SEQ ID NO: 216 Light KappaDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 217Heavy IgG1 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVS V I S SQGKSTYYADSVKGRFTISRDENSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGKMOR10703 A50V_N52G SEQ ID NO: 218 (Kabat) HCDR1 SYAMSSEQ ID NO: 219 (Kabat) HCDR2 V I G SQGKSTYYADSVKG SEQ ID NO: 220 (Kabat)HCDR3 WGDEGFDI SEQ ID NO: 221 (Kabat) LCDR1 RASQGISNWLASEQ ID NO: 222 (Kabat) LCDR2 GASSLQS SEQ ID NO: 223 (Kabat) LCDR3QQYSSFPTT SEQ ID NO: 224 (Chothia) HCDR1 GFTFSSYSEQ ID NO: 225 (Chothia) HCDR2 G SQGKS SEQ ID NO: 226 (Chothia) HCDR3WGDEGFDI SEQ ID NO: 227 (Chothia) LCDR1 SQGISNW SEQ ID NO: 228 (Chothia)LCDR2 GAS SEQ ID NO: 229 (Chothia) LCDR3 YSSFPT SEQ ID NO: 230 VLDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTTFGQGTKVEIK SEQ ID NO: 231 VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVS V I G SQGKSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDIWGQG TLVTVSSSEQ ID NO: 232 DNA VLGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGAGTGACCATCACCTGTCGGGCCAGCCAGGGCATCAGCAACTGGCTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGGCGCCAGCTCCCTGCAGAGCGGCGTGCCAAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAGCAGCTTCCCCACCACCTTCGGCCAGGGCACCAAGGTGGAAATCAAG SEQ ID NO: 233 DNA VHGAGGTGCAATTGCTGGAAAGCGGCGGAGGCCTGGTGCAGCCTGGCGGCAGCCTGAGACTGTCTTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTCCGCCAGGCCCCTGGCAAGGGACTGGAATGGGTGTCCGTCATCGGCAGCCAGGGCAAGAGCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTGCCAGATGGGGCGACGAGGGCTTCGACATCTGGGGCCAGGGCACCCTGGTCACCGTCAGCTCA SEQ ID NO: 234 Light KappaDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 235Heavy IgG1 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVS V I G SQGKSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGKMOR10703 S52aA SEQ ID NO: 236 (Kabat) HCDR1 SYAMS SEQ ID NO: 237 (Kabat)HCDR2 AIN A QGKSTYYADSVKG SEQ ID NO: 238 (Kabat) HCDR3 WGDEGFDISEQ ID NO: 239 (Kabat) LCDR1 RASQGISNWLA SEQ ID NO: 240 (Kabat) LCDR2GASSLQS SEQ ID NO: 241 (Kabat) LCDR3 QQYSSFPTT SEQ ID NO: 242 (Chothia)HCDR1 GFTFSSY SEQ ID NO: 243 (Chothia) HCDR2 N A QGKSSEQ ID NO: 244 (Chothia) HCDR3 WGDEGFDI SEQ ID NO: 245 (Chothia) LCDR1SQGISNW SEQ ID NO: 246 (Chothia) LCDR2 GAS SEQ ID NO: 247 (Chothia)LCDR3 YSSFPT SEQ ID NO: 248 VLDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTTFGQGTKVEIK SEQ ID NO: 249 VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIN A QGKSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDIWGQG TLVTVSSSEQ ID NO: 250 DNA VLGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGAGTGACCATCACCTGTCGGGCCAGCCAGGGCATCAGCAACTGGCTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGGCGCCAGCTCCCTGCAGAGCGGCGTGCCAAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAGCAGCTTCCCCACCACCTTCGGCCAGGGCACCAAGGTGGAAATCAAG SEQ ID NO: 251 DNAVHGAGGTGCAATTGCTGGAAAGCGGCGGAGGCCTGGTGCAGCCTGGCGGCAGCCTGAGACTGTCTTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTCCGCCAGGCCCCTGGCAAGGGACTGGAATGGGTGTCCGCCATCAACGCCCAGGGCAAGAGCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTGCCAGATGGGGCGACGAGGGCTTCGACATCTGGGGCCAGGGCACCCTGGTCACCGTCAGCTCA SEQ ID NO: 252 Light KappaDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 253Heavy IgG1 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIN A QGKSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGKMOR10703 S52aT SEQ ID NO: 254 (Kabat) HCDR1 SYAMS SEQ ID NO: 255 (Kabat)HCDR2 AIN T QGKSTYYADSVKG SEQ ID NO: 256 (Kabat) HCDR3 WGDEGFDISEQ ID NO: 257 (Kabat) LCDR1 RASQGISNWLA SEQ ID NO: 258 (Kabat) LCDR2GASSLQS SEQ ID NO: 259 (Kabat) LCDR3 QQYSSFPTT SEQ ID NO: 260 (Chothia)HCDR1 GFTFSSY SEQ ID NO: 261 (Chothia) HCDR2 N T QGKSSEQ ID NO: 262 (Chothia) HCDR3 WGDEGFDI SEQ ID NO: 263 (Chothia) LCDR1SQGISNW SEQ ID NO: 264 (Chothia) LCDR2 GAS SEQ ID NO: 265 (Chothia)LCDR3 YSSFPT SEQ ID NO: 266 VLDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTTFGQGTKVEIK SEQ ID NO: 267 VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIN T QGKSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDIWGQG TLVTVSSSEQ ID NO: 268 DNA VLGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGAGTGACCATCACCTGTCGGGCCAGCCAGGGCATCAGCAACTGGCTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGGCGCCAGCTCCCTGCAGAGCGGCGTGCCAAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAGCAGCTTCCCCACCACCTTCGGCCAGGGCACCAAGGTGGAAATCAAG SEQ ID NO: 269 DNA VHGAGGTGCAATTGCTGGAAAGCGGCGGAGGCCTGGTGCAGCCTGGCGGCAGCCTGAGACTGTCTTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTCCGCCAGGCCCCTGGCAAGGGACTGGAATGGGTGTCCGCCATCAACACCCAGGGCAAGAGCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTGCCAGATGGGGCGACGAGGGCTTCGACATCTGGGGCCAGGGCACCCTGGTCACCGTCAGCTCA SEQ ID NO: 270 Light KappaDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 271Heavy IgG1 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIN T QGKSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGKMOR10701 R55S SEQ ID NO: 272 (Kabat) HCDR1 SYAMS SEQ ID NO: 273 (Kabat)HCDR2 VTGAVG S STYYPDSVKG SEQ ID NO: 274 (Kabat) HCDR3 WGDEGFDISEQ ID NO: 275 (Kabat) LCDR1 RASQGISNWLA SEQ ID NO: 276 (Kabat) LCDR2GASSLQS SEQ ID NO: 277 (Kabat) LCDR3 QQYSSFPTT SEQ ID NO: 278 (Chothia)HCDR1 GFTFSSY SEQ ID NO: 279 (Chothia) HCDR2 GAVG S SSEQ ID NO: 280 (Chothia) HCDR3 WGDEGFDI SEQ ID NO: 281 (Chothia) LCDR1SQGISNW SEQ ID NO: 282 (Chothia) LCDR2 GAS SEQ ID NO: 283 (Chothia)LCDR3 YSSFPT SEQ ID NO: 284 VLDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTTFGQGTKVEIK SEQ ID NO: 285 VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVTGAVG SSTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDIWGQG TLVTVSSSEQ ID NO: 286 DNA VLGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGAGTGACCATCACCTGTCGGGCCAGCCAGGGCATCAGCAACTGGCTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGGCGCCAGCTCCCTGCAGAGCGGCGTGCCAAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAGCAGCTTCCCCACCACCTTCGGCCAGGGCACCAAGGTGGAAATCAAG SEQ ID NO: 287 DNA VHGAGGTGCAATTGCTGGAAAGCGGCGGAGGCCTGGTGCAGCCTGGCGGCAGCCTGAGACTGTCTTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTCCGCCAGGCCCCTGGCAAGGGACTGGAATGGGTGTCCGTGACAGGCGCCGTGGGCAGCAGCACCTACTACCCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTGCCAGATGGGGCGACGAGGGCTTCGACATCTGGGGCCAGGGCACCCTGGTCACCGTCAGCTCA SEQ ID NO: 288 Light KappaDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 289Heavy IgG1 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVTGAVG SSTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGKMOR10701 R55G SEQ ID NO: 290 (Kabat) HCDR1 SYAMS SEQ ID NO: 291 (Kabat)HCDR2 VTGAVG G STYYPDSVKG SEQ ID NO: 292 (Kabat) HCDR3 WGDEGFDISEQ ID NO: 293 (Kabat) LCDR1 RASQGISNWLA SEQ ID NO: 294 (Kabat) LCDR2GASSLQS SEQ ID NO: 295 (Kabat) LCDR3 QQYSSFPTT SEQ ID NO: 296 (Chothia)HCDR1 GFTFSSY SEQ ID NO: 297 (Chothia) HCDR2 GAVG G SSEQ ID NO: 298 (Chothia) HCDR3 WGDEGFDI SEQ ID NO: 299 (Chothia) LCDR1SQGISNW SEQ ID NO: 300 (Chothia) LCDR2 GAS SEQ ID NO: 301 (Chothia)LCDR3 YSSFPT SEQ ID NO: 302 VLDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTTFGQGTKVEIK SEQ ID NO: 303 VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVTGAVG GSTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDIWGQG TLVTVSSSEQ ID NO: 304 DNA VLGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGAGTGACCATCACCTGTCGGGCCAGCCAGGGCATCAGCAACTGGCTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGGCGCCAGCTCCCTGCAGAGCGGCGTGCCAAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAGCAGCTTCCCCACCACCTTCGGCCAGGGCACCAAGGTGGAAATCAAG SEQ ID NO: 305 DNA VHGAGGTGCAATTGCTGGAAAGCGGCGGAGGCCTGGTGCAGCCTGGCGGCAGCCTGAGACTGTCTTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTCCGCCAGGCCCCTGGCAAGGGACTGGAATGGGTGTCCGTGACAGGCGCCGTGGGCGGAAGCACCTACTACCCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTGCCAGATGGGGCGACGAGGGCTTCGACATCTGGGGCCAGGGCACCCTGGTCACCGTCAGCTCA SEQ ID NO: 306 Light KappaDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 307Heavy IgG1 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVTGAVG GSTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGKMOR10701 R55K SEQ ID NO: 308 (Kabat) HCDR1 SYAMS SEQ ID NO: 309 (Kabat)HCDR2 VTGAVG K STYYPDSVKG SEQ ID NO: 310 (Kabat) HCDR3 WGDEGFDISEQ ID NO: 311 (Kabat) LCDR1 RASQGISNWLA SEQ ID NO: 312 (Kabat) LCDR2GASSLQS SEQ ID NO: 313 (Kabat) LCDR3 QQYSSFPTT SEQ ID NO: 314 (Chothia)HCDR1 GFTFSSY SEQ ID NO: 315 (Chothia) HCDR2 GAVG K SSEQ ID NO: 316 (Chothia) HCDR3 WGDEGFDI SEQ ID NO: 317 (Chothia) LCDR1SQGISNW SEQ ID NO: 318 (Chothia) LCDR2 GAS SEQ ID NO: 319 (Chothia)LCDR3 YSSFPT SEQ ID NO: 320 VLDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTTFGQGTKVEIK SEQ ID NO: 321 VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVTGAVG KSTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDIWGQG TLVTVSSSEQ ID NO: 322 DNA VLGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGAGTGACCATCACCTGTCGGGCCAGCCAGGGCATCAGCAACTGGCTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGGCGCCAGCTCCCTGCAGAGCGGCGTGCCAAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAGCAGCTTCCCCACCACCTTCGGCCAGGGCACCAAGGTGGAAATCAAG SEQ ID NO: 323 DNA VHGAGGTGCAATTGCTGGAAAGCGGCGGAGGCCTGGTGCAGCCTGGCGGCAGCCTGAGACTGTCTTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTCCGCCAGGCCCCTGGCAAGGGACTGGAATGGGTGTCCGTGACAGGCGCCGTGGGCAAAAGCACCTACTACCCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTGCCAGATGGGGCGACGAGGGCTTCGACATCTGGGGCCAGGGCACCCTGGTCACCGTCAGCTCA SEQ ID NO: 324 Light KappaDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 325Heavy IgG1 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVTGAVG KSTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGKMOR10701 deletion S56 SEQ ID NO: 326 (Kabat) HCDR1 SYAMSSEQ ID NO: 327 (Kabat) HCDR2 VTGAVGRTYYPDSVKG SEQ ID NO: 328 (Kabat)HCDR3 WGDEGFDI SEQ ID NO: 329 (Kabat) LCDR1 RASQGISNWLASEQ ID NO: 330 (Kabat) LCDR2 GASSLQS SEQ ID NO: 331 (Kabat) LCDR3QQYSSFPT SEQ ID NO: 332 (Chothia) HCDR1 GFTFSSY SEQ ID NO: 333 (Chothia)HCDR2 GAVGRT SEQ ID NO: 334 (Chothia) HCDR3 WGDEGFDISEQ ID NO: 335 (Chothia) LCDR1 SQGISNW SEQ ID NO: 336 (Chothia) LCDR2GAS SEQ ID NO: 337 (Chothia) LCDR3 YSSFPT SEQ ID NO: 338 VLDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTTFGQGTKVEIK SEQ ID NO: 339 VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVTGAVGRTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDIWGQGT LVTVSSSEQ ID NO: 340 DNA VLGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGAGTGACCATCACCTGTCGGGCCAGCCAGGGCATCAGCAACTGGCTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGGCGCCAGCTCCCTGCAGAGCGGCGTGCCAAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAGCAGCTTCCCCACCACCTTCGGCCAGGGCACCAAGGTGGAAATCAAG SEQ ID NO: 341 DNA VHGAGGTGCAATTGCTGGAAAGCGGCGGAGGCCTGGTGCAGCCTGGCGGCAGCCTGAGACTGTCTTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTCCGCCAGGCCCCTGGCAAGGGACTGGAATGGGTGTCCGTGACAGGCGCCGTGGGCAGAACCTACTACCCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTGCCAGATGGGGCGACGAGGGCTTCGACATCTGGGGCCAGGGCACCCTGGTCACCGTCAGCTCA SEQ ID NO: 342 Light KappaDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 343Heavy IgG1 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVTGAVGRTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK MOR12609SEQ ID NO: 344 (Kabat) HCDR1 SYAMS SEQ ID NO: 345 (Kabat) HCDR2VINGLGYTTFYADSVKG SEQ ID NO: 346 (Kabat) HCDR3 WGDEGFDISEQ ID NO: 347 (Kabat) LCDR1 RASQGISNWLA SEQ ID NO: 348 (Kabat) LCDR2GASSLQS SEQ ID NO: 349 (Kabat) LCDR3 QQYSSFPTT SEQ ID NO: 350 (Chothia)HCDR1 GFTFSSY SEQ ID NO: 351 (Chothia) HCDR2 NGLGYTSEQ ID NO: 352 (Chothia) HCDR3 WGDEGFDI SEQ ID NO: 353 (Chothia) LCDR1SQGISNW SEQ ID NO: 354 (Chothia) LCDR2 GAS SEQ ID NO: 355 (Chothia)LCDR3 YSSFPT SEQ ID NO: 356 VLDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQYSSFPTTFGQGTKVEIK SEQ ID NO: 357 VHQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVINGLGYTTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDIWGQG TLVTVSSSEQ ID NO: 358 DNA VLGATATCCAGATGACCCAGAGCCCGTCTAGCCTGAGCGCGAGCGTGGGTGATCGTGTGACCATTACCTGCAGAGCGAGCCAGGGTATTTCTAATTGGCTGGCTTGGTACCAGCAGAAACCAGGTAAAGCACCGAAACTATTAATTTATGGTGCTTCTTCTTTGCAAAGCGGGGTCCCGTCCCGTTTTAGCGGCTCTGGATCCGGCACTGATTTTACCCTGACCATTAGCAGCCTGCAACCTGAAGACTTTGCGGTTTATTATTGCCAGCAGTATTCTTCTTTTCCTACTACCTTTGGCCAGGGTACGAAAGTTGAAATTAAA SEQ ID NO: 359 DNA VHCAGGTGCAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCCTGCGTCTGAGCTGCGCGGCCTCCGGATTTACCTTTAGCAGCTATGCGATGAGCTGGGTGCGCCAAGCCCCTGGGAAGGGTCTCGAGTGGGTGAGCGTTATTAATGGTCTTGGTTATACTACTTTTTATGCTGATTCTGTTAAGGGTCGTTTTACCATTTCACGTGATAATTCGAAAAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGCGCGTTGGGGTGATGAGGGTTTTGATATTTGGGGCCAAGGCACCCTGGTGACGGTTAGCTCA SEQ ID NO: 360 Light KappaDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQYSSFPTTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 361Heavy IgG1 QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVINGLGYTTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK MOR12610SEQ ID NO: 362 (Kabat) HCDR1 SYAMS SEQ ID NO: 363 (Kabat) HCDR2GTGPYGGTYYPDSVKG SEQ ID NO: 364 (Kabat) HCDR3 WGDEGFDISEQ ID NO: 365 (Kabat) LCDR1 RASQGISNWLA SEQ ID NO: 366 (Kabat) LCDR2GASSLQS SEQ ID NO: 367 (Kabat) LCDR3 QQYSSFPTT SEQ ID NO: 368 (Chothia)HCDR1 GFTFSSY SEQ ID NO: 369 (Chothia) HCDR2 GPYGGSEQ ID NO: 370 (Chothia) HCDR3 WGDEGFDI SEQ ID NO: 371 (Chothia) LCDR1SQGISNW SEQ ID NO: 372 (Chothia) LCDR2 GAS SEQ ID NO: 373 (Chothia)LCDR3 YSSFPT SEQ ID NO: 374 VLDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQYSSFPTTFGQGTKVEIK SEQ ID NO: 375 VHQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGTGPYGGTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDIWGQGT LVTVSSSEQ ID NO: 376 DNA VLGATATCCAGATGACCCAGAGCCCGTCTAGCCTGAGCGCGAGCGTGGGTGATCGTGTGACCATTACCTGCAGAGCGAGCCAGGGTATTTCTAATTGGCTGGCTTGGTACCAGCAGAAACCAGGTAAAGCACCGAAACTATTAATTTATGGTGCTTCTTCTTTGCAAAGCGGGGTCCCGTCCCGTTTTAGCGGCTCTGGATCCGGCACTGATTTTACCCTGACCATTAGCAGCCTGCAACCTGAAGACTTTGCGGTTTATTATTGCCAGCAGTATTCTTCTTTTCCTACTACCTTTGGCCAGGGTACGAAAGTTGAAATTAAA SEQ ID NO: 377 DNA VHCAGGTGCAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCCTGCGTCTGAGCTGCGCGGCCTCCGGATTTACCTTTAGCAGCTATGCGATGAGCTGGGTGCGCCAAGCCCCTGGGAAGGGTCTCGAGTGGGTGAGCGGTACTGGTCCTTATGGTGGTACTTATTATCCTGATTCTGTTAAGGGTCGTTTTACCATTTCACGTGATAATTCGAAAAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGCGCGTTGGGGTGATGAGGGTTTTGATATTTGGGGCCAAGGCACCCTGGTGACGGTTAGCTCA SEQ ID NO: 378 Light KappaDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQYSSFPTTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 379Heavy IgG1 QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGTGPYGGTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK

Other antibodies of the invention include those where the amino acids ornucleic acids encoding the amino acids have been mutated, yet have atleast 60, 70, 80, 90, 95, 96, 97, 98, and 99 percent identity to thesequences described in Table 1. In some embodiments, it include mutantamino acid sequences wherein no more than 1, 2, 3, 4 or 5 amino acidshave been mutated in the variable regions when compared with thevariable regions depicted in the sequence described in Table 1, whileretaining substantially the same therapeutic activity.

Since each of these antibodies or fragments thereof can bind to HER3,the VH, VL, full length light chain, and full length heavy chainsequences (amino acid sequences and the nucleotide sequences encodingthe amino acid sequences) can be “mixed and matched” to create otherHER3-binding antibodies of the invention. Such “mixed and matched”HER3-binding antibodies can be tested using the binding assays known inthe art (e.g., ELISAs, and other assays described in the Examplesection). When these chains are mixed and matched, a VH sequence from aparticular VH/VL pairing should be replaced with a structurally similarVH sequence. Likewise a full length heavy chain sequence from aparticular full length heavy chain/full length light chain pairingshould be replaced with a structurally similar full length heavy chainsequence. Likewise, a VL sequence from a particular VH/VL pairing shouldbe replaced with a structurally similar VL sequence. Likewise a fulllength light chain sequence from a particular full length heavychain/full length light chain pairing should be replaced with astructurally similar full length light chain sequence. Accordingly, inone aspect, the invention provides an isolated monoclonal antibody orfragment thereof having: a heavy chain variable region comprising anamino acid sequence selected from the group consisting of SEQ ID NO: 15,33, 51, 69, 87, 105, 123, 141, 159, 177, 195, 213, 231, 249, 267, 285,303, 321, 339, 357, and 375; and a light chain variable regioncomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 14, 32, 50, 68, 86, 104, 122, 140, 158, 176, 194, 212, 230,248, 266, 284, 302, 320, 338, 356, and 374; wherein the antibodyspecifically binds to HER3 (e.g., human and/or cynomologus).

In another aspect, the present invention provides HER3-bindingantibodies that comprise the heavy chain and light chain CDR1s, CDR2sand CDR3s as described in Table 1, or combinations thereof. The aminoacid sequences of the VH CDR1s of the antibodies are shown in SEQ IDNOs: 2, 8, 20, 26, 38, 44, 56, 62, 74, 80, 92, 98, 110, 116, 128, 134,146, 152, 164, 170, 182, 188, 200, 206, 218, 224, 236, 242, 254, 260,272, 278, 290, 296, 308, 314, 326, 332, 344, 350, 362, and 368. Theamino acid sequences of the VH CDR2s of the antibodies and are shown inSEQ ID NOs: 3, 9, 21, 27, 39, 45, 57, 63, 75, 81, 93, 99, 111,117,129,135,147,153,165,171,183,189,201,207,219,225,237,243,255,261,273,279,291, 297, 309, 315, 327, 333, 345, 351, 363, and 369. The amino acidsequences of the VH CDR3s of the antibodies are shown in SEQ ID NOs: 4,10, 22, 28, 40, 46, 58, 64, 76, 82, 94, 100,112,118,130,136,148,154,166, 172, 184, 190,202,208,220,226,238,244,256,262, 274, 280, 292, 298,310, 316, 328, 334, 346, 352, 364, and 370. The amino acid sequences ofthe VL CDR1s of the antibodies are shown in SEQ ID NOs: 5, 11, 23, 29,41, 47, 59, 65, 77, 83, 95, 101, 113, 119, 131, 137, 149, 155, 167, 173,185, 191, 203, 209, 221, 227, 239, 245, 257, 263, 275, 281, 293, 299,311, 317, 329, 335, 347, 353, 365, and 371. The amino acid sequences ofthe VL CDR2s of the antibodies are shown in SEQ ID NOs: 6, 12, 24, 30,42, 48, 60, 66, 78, 84, 96, 102, 114, 120, 132, 138, 150, 156, 168, 174,186, 192, 204, 210, 222, 228, 240, 246, 258, 264, 276, 282, 294, 300,312, 318, 330, 336, 348, 354, 366, and 372. The amino acid sequences ofthe VL CDR3s of the antibodies are shown in SEQ ID NOs: 7, 13, 25, 31,43, 49, 61, 67, 79, 85, 97, 103, 115, 121, 133, 139, 151, 157, 169, 175,187, 193, 205, 211, 223, 229, 241, 247, 259, 265, 277, 283, 295, 301,313, 319, 331, 337, 349, 355, 367, and 373. The CDR regions aredelineated using the Kabat system (Kabat et al., (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242; Chothia et al.,(1987) J. Mol. Biol. 196:901-917; Chothia et al., (1989) Nature 342:877-883; and Al-Lazikani et al., (1997) J. Mol. Biol. 273, 927-948).

In a specific embodiment, an antibody that binds to HER3 comprises aheavy chain variable region CDR1 of SEQ ID NO: 2; a CDR2 of SEQ ID NO:3; a CDR3 of SEQ ID NO: 4; a light chain variable region CDR1 of SEQ IDNO: 5; a CDR2 of SEQ ID NO: 6; and a CDR3 of SEQ ID NO: 7.

In a specific embodiment, an antibody that binds to HER3 comprises aheavy chain variable region CDR1 of SEQ ID NO: 20; a CDR2 of SEQ ID NO:21; a CDR3 of SEQ ID NO: 22; a light chain variable region CDR1 of SEQID NO: 23; a CDR2 of SEQ ID NO: 24; and a CDR3 of SEQ ID NO: 25.

In a specific embodiment, an antibody that binds to HER3 comprises aheavy chain variable region CDR1 of SEQ ID NO: 38; a CDR2 of SEQ ID NO:39; a CDR3 of SEQ ID NO: 40; a light chain variable region CDR1 of SEQID NO: 41; a CDR2 of SEQ ID NO: 42; and a CDR3 of SEQ ID NO: 43.

In a specific embodiment, an antibody that binds to HER3 comprises aheavy chain variable region CDR1 of SEQ ID NO: 56; a CDR2 of SEQ ID NO:57; a CDR3 of SEQ ID NO: 58; a light chain variable region CDR1 of SEQID NO: 59; a CDR2 of SEQ ID NO: 60; and a CDR3 of SEQ ID NO: 61.

In a specific embodiment, an antibody that binds to HER3 comprises aheavy chain variable region CDR1 of SEQ ID NO: 74; a CDR2 of SEQ ID NO:75; a CDR3 of SEQ ID NO: 76; a light chain variable region CDR1 of SEQID NO: 77; a CDR2 of SEQ ID NO: 78; and a CDR3 of SEQ ID NO: 79.

In a specific embodiment, an antibody that binds to HER3 comprises aheavy chain variable region CDR1 of SEQ ID NO: 92; a CDR2 of SEQ ID NO:93; a CDR3 of SEQ ID NO: 94; a light chain variable region CDR1 of SEQID NO: 95; a CDR2 of SEQ ID NO: 96; and a CDR3 of SEQ ID NO: 97.

In a specific embodiment, an antibody that binds to HER3 comprises aheavy chain variable region CDR1 of SEQ ID NO: 110; a CDR2 of SEQ ID NO:111; a CDR3 of SEQ ID NO: 112; a light chain variable region CDR1 of SEQID NO: 113; a CDR2 of SEQ ID NO: 114; and a CDR3 of SEQ ID NO: 115.

In a specific embodiment, an antibody that binds to HER3 comprises aheavy chain variable region CDR1 of SEQ ID NO: 128; a CDR2 of SEQ ID NO:129; a CDR3 of SEQ ID NO: 130; a light chain variable region CDR1 of SEQID NO: 131; a CDR2 of SEQ ID NO: 132; and a CDR3 of SEQ ID NO: 133.

In a specific embodiment, an antibody that binds to HER3 comprises aheavy chain variable region CDR1 of SEQ ID NO: 146; a CDR2 of SEQ ID NO:147; a CDR3 of SEQ ID NO: 148; a light chain variable region CDR1 of SEQID NO: 149; a CDR2 of SEQ ID NO: 150; and a CDR3 of SEQ ID NO: 151.

In a specific embodiment, an antibody that binds to HER3 comprises aheavy chain variable region CDR1 of SEQ ID NO: 164; a CDR2 of SEQ ID NO:165; a CDR3 of SEQ ID NO: 166; a light chain variable region CDR1 of SEQID NO: 167; a CDR2 of SEQ ID NO: 168; and a CDR3 of SEQ ID NO: 169.

In a specific embodiment, an antibody that binds to HER3 comprises aheavy chain variable region CDR1 of SEQ ID NO: 182; a CDR2 of SEQ ID NO:183; a CDR3 of SEQ ID NO: 184; a light chain variable region CDR1 of SEQID NO: 185; a CDR2 of SEQ ID NO: 186; and a CDR3 of SEQ ID NO: 187.

In a specific embodiment, an antibody that binds to HER3 comprises aheavy chain variable region CDR1 of SEQ ID NO: 200; a CDR2 of SEQ ID NO:201; a CDR3 of SEQ ID NO: 202; a light chain variable region CDR1 of SEQID NO: 203; a CDR2 of SEQ ID NO: 204; and a CDR3 of SEQ ID NO: 205.

In a specific embodiment, an antibody that binds to HER3 comprises aheavy chain variable region CDR1 of SEQ ID NO: 218; a CDR2 of SEQ ID NO:219; a CDR3 of SEQ ID NO: 220; a light chain variable region CDR1 of SEQID NO: 221; a CDR2 of SEQ ID NO: 222; and a CDR3 of SEQ ID NO: 223.

In a specific embodiment, an antibody that binds to HER3 comprises aheavy chain variable region CDR1 of SEQ ID NO: 236; a CDR2 of SEQ ID NO:237; a CDR3 of SEQ ID NO: 238; a light chain variable region CDR1 of SEQID NO: 239; a CDR2 of SEQ ID NO: 240; and a CDR3 of SEQ ID NO: 241.

In a specific embodiment, an antibody that binds to HER3 comprises aheavy chain variable region CDR1 of SEQ ID NO: 254; a CDR2 of SEQ ID NO:255; a CDR3 of SEQ ID NO: 256; a light chain variable region CDR1 of SEQID NO: 257; a CDR2 of SEQ ID NO: 258; and a CDR3 of SEQ ID NO: 259.

In a specific embodiment, an antibody that binds to HER3 comprises aheavy chain variable region CDR1 of SEQ ID NO: 272; a CDR2 of SEQ ID NO:273; a CDR3 of SEQ ID NO: 274; a light chain variable region CDR1 of SEQID NO: 275; a CDR2 of SEQ ID NO: 276; and a CDR3 of SEQ ID NO: 277.

In a specific embodiment, an antibody that binds to HER3 comprises aheavy chain variable region CDR1 of SEQ ID NO: 290; a CDR2 of SEQ ID NO:291; a CDR3 of SEQ ID NO: 292; a light chain variable region CDR1 of SEQID NO: 293; a CDR2 of SEQ ID NO: 294; and a CDR3 of SEQ ID NO: 295.

In a specific embodiment, an antibody that binds to HER3 comprises aheavy chain variable region CDR1 of SEQ ID NO: 308; a CDR2 of SEQ ID NO:309; a CDR3 of SEQ ID NO: 310; a light chain variable region CDR1 of SEQID NO: 311; a CDR2 of SEQ ID NO: 312; and a CDR3 of SEQ ID NO: 313.

In a specific embodiment, an antibody that binds to HER3 comprises aheavy chain variable region CDR1 of SEQ ID NO: 326; a CDR2 of SEQ ID NO:327; a CDR3 of SEQ ID NO: 328; a light chain variable region CDR1 of SEQID NO: 329; a CDR2 of SEQ ID NO: 330; and a CDR3 of SEQ ID NO: 331.

In a specific embodiment, an antibody that binds to HER3 comprises aheavy chain variable region CDR1 of SEQ ID NO: 344; a CDR2 of SEQ ID NO:345; a CDR3 of SEQ ID NO: 346; a light chain variable region CDR1 of SEQID NO: 347; a CDR2 of SEQ ID NO: 348; and a CDR3 of SEQ ID NO: 349.

In a specific embodiment, an antibody that binds to HER3 comprises aheavy chain variable region CDR1 of SEQ ID NO: 362; a CDR2 of SEQ ID NO:363; a CDR3 of SEQ ID NO: 364; a light chain variable region CDR1 of SEQID NO: 365; a CDR2 of SEQ ID NO: 366; and a CDR3 of SEQ ID NO: 367.

In a specific embodiment, an antibody that binds to HER3 comprises a VHof SEQ ID NO. 15 and VL of SEQ ID NO: 14. In a specific embodiment, anantibody that binds to HER3 comprises a VH of SEQ ID NO: 33 and VL ofSEQ ID NO: 32. In a specific embodiment, an antibody that binds to HER3comprises a VH of SEQ ID NO: 51 and VL of SEQ ID NO: 50. In a specificembodiment, an antibody that binds to HER3 comprises a SEQ ID NO: 69 andVL of SEQ ID NO: 68. In a specific embodiment, an antibody that binds toHER3 comprises a VH of SEQ ID NO: 87 and VL of SEQ ID NO: 86. In aspecific embodiment, an antibody that binds to HER3 comprises a VH ofSEQ ID NO: 105 and VL of SEQ ID NO: 104. In a specific embodiment, anantibody that binds to HER3 comprises a VH of SEQ ID NO: 123 and VL ofSEQ ID NO: 122. In a specific embodiment, an antibody that binds to HER3comprises a VH of SEQ ID NO: 141 and VL of SEQ ID NO: 140. In a specificembodiment, an antibody that binds to HER3 comprises a VH of SEQ ID NO:159 and VL of SEQ ID NO: 158. In a specific embodiment, an antibody thatbinds to HER3 comprises a VH of SEQ ID NO: 177 and VL of SEQ ID NO: 176.In a specific embodiment, an antibody that binds to HER3 comprises a VHof SEQ ID NO: 195 and VL of SEQ ID NO: 194. In a specific embodiment, anantibody that binds to HER3 comprises a VH of SEQ ID NO: 213 and VL ofSEQ ID NO: 212. In a specific embodiment, an antibody that binds to HER3comprises a VH of SEQ ID NO: 231 and VL of SEQ ID NO: 230. In a specificembodiment, an antibody that binds to HER3 comprises a VH of SEQ ID NO:249 and VL of SEQ ID NO: 248. In a specific embodiment, an antibody thatbinds to HER3 comprises a VH of SEQ ID NO: 267 and VL of SEQ ID NO: 266.In a specific embodiment, an antibody that binds to HER3 comprises a VHof SEQ ID NO: 285 and VL of SEQ ID NO: 284. In a specific embodiment, anantibody that binds to HER3 comprises a VH of SEQ ID NO: 303 and VL ofSEQ ID NO: 302. In a specific embodiment, an antibody that binds to HER3comprises a VH of SEQ ID NO: 321 and VL of SEQ ID NO: 320. In a specificembodiment, an antibody that binds to HER3 comprises a VH of SEQ ID NO:339 and VL of SEQ ID NO: 338. In a specific embodiment, an antibody thatbinds to HER3 comprises a VH of SEQ ID NO: 357 and VL of SEQ ID NO: 356.In a specific embodiment, an antibody that binds to HER3 comprises a VHof SEQ ID NO: 375 and VL of SEQ ID NO: 374. In one embodiment, the HER3antibodies are antagonist antibodies. In certain embodiments, anantibody that binds to HER3 is an antibody that is described in Table 1.

As used herein, a human antibody comprises heavy or light chain variableregions or full length heavy or light chains that are “the product of”or “derived from” a particular germline sequence if the variable regionsor full length chains of the antibody are obtained from a system thatuses human germline immunoglobulin genes. Such systems includeimmunizing a transgenic mouse carrying human immunoglobulin genes withthe antigen of interest or screening a human immunoglobulin gene librarydisplayed on phage with the antigen of interest. A human antibody thatis “the product of” or “derived from” a human germline immunoglobulinsequence can be identified as such by comparing the amino acid sequenceof the human antibody to the amino acid sequences of human germlineimmunoglobulins and selecting the human germline immunoglobulin sequencethat is closest in sequence (i.e., greatest % identity) to the sequenceof the human antibody. A human antibody that is “the product of” or“derived from” a particular human germline immunoglobulin sequence maycontain amino acid differences as compared to the germline sequence, dueto, for example, naturally occurring somatic mutations or intentionalintroduction of site-directed mutations. However, in the VH or VLframework regions, a selected human antibody typically is at least 90%identical in amino acids sequence to an amino acid sequence encoded by ahuman germline immunoglobulin gene and contains amino acid residues thatidentify the human antibody as being human when compared to the germlineimmunoglobulin amino acid sequences of other species (e.g., murinegermline sequences). In certain cases, a human antibody may be at least60%, 70%, 80%, 90%, or at least 95%, or even at least 96%, 97%, 98%, or99% identical in amino acid sequence to the amino acid sequence encodedby the germline immunoglobulin gene. Typically, a recombinant humanantibody will display no more than 10 amino acid differences from theamino acid sequence encoded by the human germline immunoglobulin gene inthe VH or VL framework regions. In certain cases, the human antibody maydisplay no more than 5, or even no more than 4, 3, 2, or 1 amino aciddifference from the amino acid sequence encoded by the germlineimmunoglobulin gene. Different germlined versions using the VH and VLgermline sequences for a representative number of HER3 antibodies isshown in Table 2, using Kabat. The CDR positions are highlighted inboldface. The notation used in the Tables with germlined sequences is asfollows: MOR10701-VH_3-07 means MOR10701 CDR loops in framework regionsof VH germline sequence 3-07 (nomenclature is according to Vbase),MOR10703-VK_L1 means CDR from MOR10703 in germline framework regionsfrom VK_L1, where VK is the kappa light chain.

TABLE 2Different germlined versions of a selected number of representative antibodiesSEQ ID NUMBER Sequence Name Amino Acid Sequence MOR10701 VH domainSEQ ID NO: 380 MOR10701-VH_3-07EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVAVTGAVGRSTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARWGDEGFDI SEQ ID NO: 381MOR10701-VH_3-09EVQLVESGGGLVQPGRSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVTGAVGRSTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKWGDEGFDI SEQ ID NO: 382MOR10701-VH_3-11QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWIRQAPGKGLEWVSVTGAVGRSTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARWGDEGFDI SEQ ID NO: 383MOR10701-VH_3-13EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQATGKGLEWVSVTGAVGRSTYYPDSVKGRFTISRENAKNSLYLQMNSLRAGDTAVYYCARWGDEGFDI SEQ ID NO: 384MOR10701-VH_3-15EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVGVTGAVGRSTYYPDSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTWGDEGFDI SEQ ID NO: 385MOR10701-VH_3-20EVQLVESGGGVVRPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVTGAVGRSTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYHCARWGDEGFDI SEQ ID NO: 386MOR10701-VH_3-21EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVTGAVGRSTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARWGDEGFDI SEQ ID NO: 387MOR10701-VH_3-23EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVTGAVGRSTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWGDEGFDI SEQ ID NO: 388MOR10701-VH_3-30QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVAVTGAVGRSTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWGDEGFDI SEQ ID NO: 389MOR10701-VH_3-30.3QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVAVTGAVGRSTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDI SEQ ID NO: 390MOR10701-VH_3-30.5QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVAVTGAVGRSTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWGDEGFDI SEQ ID NO: 391MOR10701-VH_3-33QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVAVTGAVGRSTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDI SEQ ID NO: 392MOR10701-VH_3-43EVQLVESGGVVVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVTGAVGRSTYYPDSVKGRFTISRDNSKNSLYLQMNSLRTEDTALYYCAKWGDEGFDI SEQ ID NO: 393MOR10701-VH_3-48EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVTGAVGRSTYYPDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARWGDEGFDI SEQ ID NO: 394MOR10701-VH_3-49EVQLVESGGGLVQPGRSLRLSCTASGFTFSSYAMSWFRQAPGKGLEWVGVTGAVGRSTYYPDSVKGRFTISRDGSKSIAYLQMNSLKTEDTAVYYCTRWGDEGFDI SEQ ID NO: 395MOR10701-VH_3-53EVQLVETGGGLIQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVTGAVGRSTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDI SEQ ID NO: 396MOR10701-VH_3-64EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEYVSVTGAVGRSTYYPDSVKGRFTISRDNSKNTLYLQMGSLRAEDMAVYYCARWGDEGFDI SEQ ID NO: 397MOR10701-VH_3-66EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVTGAVGRSTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDI SEQ ID NO: 398MOR10701-VH_3-72EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVGVTGAVGRSTYYPDSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARWGDEGFDI SEQ ID NO: 399MOR10701-VH_3-73EVQLVESGGGLVQPGGSLKLSCAASGFTFSSYAMSWVRQASGKGLEWVGVTGAVGRSTYYPDSVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRWGDEGFDI SEQ ID NO: 400MOR10701-VH_3-74EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLVWVSVTGAVGRSTYYPDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARWGDEGFDI SEQ ID NO: 401MOR10701-VH_3-dEVQLVESRGVLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVTGAVGRSTYYPDSVKGRFTISRDNSKNTLHLQMNSLRAEDTAVYYCKKWGDEGFDI MOR10703 VH domainSEQ ID NO: 402 MOR10703-VH_3-07EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVAAINSQGKSTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARWGDEGFDI SEQ ID NO: 403MOR10703-VH_3-09EVQLVESGGGLVQPGRSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINSQGKSTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKWGDEGFDI SEQ ID NO: 404MOR10703-VH_3-11QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWIRQAPGKGLEWVSAINSQGKSTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARWGDEGFDI SEQ ID NO: 405MOR10703-VH_3-13EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQATGKGLEWVSAINSQGKSTYYADSVKGRFTISRENAKNSLYLQMNSLRAGDTAVYYCARWGDEGFDI SEQ ID NO: 406MOR10703-VH_3-15EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVGAINSQGKSTYYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTWGDEGFDI SEQ ID NO: 407MOR10703-VH_3-20EVQLVESGGGVVRPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINSQGKSTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYHCARWGDEGFDI SEQ ID NO: 408MOR10703-VH_3-21EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINSQGKSTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARWGDEGFDI SEQ ID NO: 409MOR10703-VH_3-23EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINSQGKSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWGDEGFDI SEQ ID NO: 410MOR10703-VH_3-30QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVAAINSQGKSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWGDEGFDI SEQ ID NO: 411MOR10703-VH_3-30.3QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVAAINSQGKSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDI SEQ ID NO: 412MOR10703-VH_3-30.5QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVAAINSQGKSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWGDEGFDI SEQ ID NO: 413MOR10703-VH_3-33QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVAAINSQGKSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDI SEQ ID NO: 414MOR10703-VH_3-43EVQLVESGGVVVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINSQGKSTYYADSVKGRFTISRDNSKNSLYLQMNSLRTEDTALYYCAKWGDEGFDI SEQ ID NO: 415MOR10703-VH_3-48EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINSQGKSTYYADSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARWGDEGFDI SEQ ID NO: 416MOR10703-VH_3-49EVQLVESGGGLVQPGRSLRLSCTASGFTFSSYAMSWFRQAPGKGLEWVGAINSQGKSTYYADSVKGRFTISRDGSKSIAYLQMNSLKTEDTAVYYCTRWGDEGFDI SEQ ID NO: 417MOR10703-VH_3-53EVQLVETGGGLIQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINSQGKSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDI SEQ ID NO: 418MOR10703-VH_3-64EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEYVSAINSQGKSTYYADSVKGRFTISRDNSKNTLYLQMGSLRAEDMAVYYCARWGDEGFDI SEQ ID NO: 419MOR10703-VH_3-66EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINSQGKSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDI SEQ ID NO: 420MOR10703-VH_3-72EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVGAINSQGKSTYYADSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARWGDEGFDI SEQ ID NO: 421MOR10703-VH_3-73EVQLVESGGGLVQPGGSLKLSCAASGFTFSSYAMSWVRQASGKGLEWVGAINSQGKSTYYADSVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRWGDEGFDI SEQ ID NO: 422MOR10703-VH_3-74EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLVWVSAINSQGKSTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARWGDEGFDI SEQ ID NO: 423MOR10703-VH_3-dEVQLVESRGVLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINSQGKSTYYADSVKGRFTISRDNSKNTLHLQMNSLRAEDTAVYYCKKWGDEGFDI MOR10701 VK domainSEQ ID NO: 424 MOR10701-VKI_O12DIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGS(same as MOR10701 GSGTDFTLTISSLQPEDFATYYCQQYSSFPTT wt) SEQ ID NO: 425MOR10701-VKI_O2DIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTT SEQ ID NO: 426 MOR10701-VKI_O18DIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYSSFPTT SEQ ID NO: 427 MOR10701-VKI_O8DIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYSSFPTT SEQ ID NO: 428 MOR10701-VKI_A20DIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKVPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQYSSFPTT SEQ ID NO: 429 MOR10701-VKI_A30DIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKRLIYGASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQYSSFPTT SEQ ID NO: 430 MOR10701-VKI_L14NIQMTQSPSAMSASVGDRVTITCRASQGISNWLAWFQQKPGKVPKHLIYGASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQYSSFPTT SEQ ID NO: 431 MOR10701-VKI_L1DIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWFQQKPGKAPKSLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTT SEQ ID NO: 432 MOR10701-VKI_L15DIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPEKAPKSLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTT SEQ ID NO: 433 MOR10701-VKI_L4AIQLTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTT SEQ ID NO: 434 MOR10701-VKI_L18AIQLTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTT SEQ ID NO: 435 MOR10701-VKI_L5DIQMTQSPSSVSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTT SEQ ID NO: 436 MOR10701-VKI_L19DIQMTQSPSSVSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTT SEQ ID NO: 437 MOR10701-VKI_L8DIQLTQSPSFLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQYSSFPTT SEQ ID NO: 438 MOR10701-VKI_L23AIRMTQSPFSLSASVGDRVTITCRASQGISNWLAWYQQKPAKAPKLFIYGASSLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYSSFPTT SEQ ID NO: 439 MOR10701-VKI_L9AIRMTQSPSSFSASTGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISCLQSEDFATYYCQQYSSFPTT SEQ ID NO: 440 MOR10701-VKI_L24VIWMTQSPSLLSASTGDRVTISCRASQGISNWLAWYQQKPGKAPELLIYGASSLQSGVPSRFSGSGSGTDFTLTISCLQSEDFATYYCQQYSSFPTT SEQ ID NO: 441 MOR10701-VKI_L11AIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTT SEQ ID NO: 442 MOR10701-VKI_L12DIQMTQSPSTLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYSSFPTT MOR10701 VK domain SEQ ID NO: 443MOR10703-VKI_O12DIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGS(same as MOR10703 GSGTDFTLTISSLQPEDFATYYCQQYSSFPTT wt) SEQ ID NO: 444MOR10703-VKI_O2DIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTT SEQ ID NO: 445 MOR10703-VKI_O18DIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYSSFPTT SEQ ID NO: 446 MOR10703-VKI_O8DIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYSSFPTT SEQ ID NO: 447 MOR10703-VKI_A20DIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKVPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQYSSFPTT SEQ ID NO: 448 MOR10703-VKI_A30DIQMTQSPSSLSASVG DRVTITCRASQGISNWLAWYQQKPGKAPKRLIYGASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQYSSFPTT SEQ ID NO: 449 MOR10703-VKI_L14NIQMTQSPSAMSASVGDRVTITCRASQGISNWLAWFQQKPGKVPKHLIYGASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQYSSFPTT SEQ ID NO: 450 MOR10703-VKI_L1DIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWFQQKPGKAPKSLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTT SEQ ID NO: 451 MOR10703-VKI_L15DIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPEKAPKSLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTT SEQ ID NO: 452 MOR10703-VKI_L4AIQLTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTT SEQ ID NO: 453 MOR10703-VKI_L18AIQLTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTT SEQ ID NO: 454 MOR10703-VKI_L5DIQMTQSPSSVSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTT SEQ ID NO: 455 MOR10703-VKI_L19DIQMTQSPSSVSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTT SEQ ID NO: 456 MOR10703-VKI_L8DIQLTQSPSFLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQYSSFPTT SEQ ID NO: 457 MOR10703-VKI_L23AIRMTQSPFSLSASVGDRVTITCRASQGISNWLAWYQQKPAKAPKLFIYGASSLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYSSFPTT SEQ ID NO: 458 MOR10703-VKI_L9AIRMTQSPSSFSASTGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISCLQSEDFATYYCQQYSSFPTT SEQ ID NO: 459 MOR10703-VKI_L24VIWMTQSPSLLSASTGDRVTISCRASQGISNWLAWYQQKPGKAPELLIYGASSLQSGVPSRFSGSGSGTDFTLTISCLQSEDFATYYCQQYSSFPTT SEQ ID NO: 460 MOR10703-VKI_L11AIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTT SEQ ID NO: 461 MOR10703-VKI_L12DIQMTQSPSTLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYSSFPTT

TABLE 3 JH segments SEQ ID NO: 462 JH1 WGQGTLVTVSS SEQ ID NO: 463 JH2WGRGTLVTVSS SEQ ID NO: 464 JH3 WGQGTMVTVSS SEQ ID NO: 465 JH4WGQGTLVTVSS SEQ ID NO: 466 JH5 WGQGTLVTVSS SEQ ID NO: 467 JH6WGQGTTVTVSS

TABLE 4 JK segments SEQ ID NO: 468 JK1 FGQGTKVEIK SEQ ID NO: 469 JK2FGQGTKLEIK SEQ ID NO: 470 JK3 FGPGTKVDIK SEQ ID NO: 471 JK4 FGGGTKVEIKSEQ ID NO: 472 JK5 FGQGTRLEIK

Any combination of the VH-germlined sequences with a JH segments can beused. Representative examples of combinations are shown in Table 5.

TABLE 5Representative examples of combinations of the VH-germlined sequences with a JHsegments. SEQ ID NO: 473 MOR10701-VH_3-EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVGVTGAVGRST 15_JH1YYPDSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTWGDEGFDIWGQGTLVTVSSSEQ ID NO: 474 MOR10701-VH_3-EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVGVTGAVGRST 15_JH3YYPDSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTWGDEGFDI WGQGTMVTVSSSEQ ID NO: 475 MOR10703-VH_3-EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVGAINSQGKSTY 15_JH1YADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTWGDEGFDIWGQGTLVTVSSSEQ ID NO: 476 MOR10703-VH_3-EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVGAINSQGKSTY 15_JH3YADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTWGDEGFDI WGQGTMVTVSS

Any combination of the VL-germlined sequences with a JK segments can beused. Representative examples of combinations are shown in Table 6.

TABLE 6Representative examples of combinations of the VK-germlined sequences with a JKsegments SEQ ID NO: 477 MOR10701-DIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSR VKI_O2_JK1FSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTTFGQGTKVEIK SEQ ID NO: 478 MOR10701-DIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYGASSLQSGVPSR VKI_O2_JK4FSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTTFGGGTKVEIK SEQ ID NO: 479 MOR10703-DIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKVPKLLIYGASSLQSGVPSRVKI_A20_JK4 FSGSGSGTDFTLTISSLQPEDVATYYCQQYSSFPTTFGGGTKVEIKSEQ ID NO: 480 MOR10703-DIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKVPKLLIYGASSLQSGVPSRVKI_A20_JK1 FSGSGSGTDFTLTISSLQPEDVATYYCQQYSSFPTTFGQGTKVEIK

Once VH has been combined with JH and VK with JK, then any combinationof VH or JH with VK or JK, can be used. In one embodiment, any of the VHgermlined regions can be combined with any of the VK (VL) germlinedregions for each antibody. A representative number of examples ofcombinations is shown in Table 7.

TABLE 7 Representative examples of combinations of germlined sequencesCombination 1 SEQ ID NO: 481 MOR10701-VH 3-EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVGVTGAVGRSTY 15_JH3YPDSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTWGDEGFDIWGQGTMVTVSSSEQ ID NO: 482 MOR10701-DIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKRLIYGASSLQSGVPSRVKI_A30_JK4 FSGSGSGTEFTLTISSLQPEDFATYYCQQYSSFPTTFGGGTKVEIK Combination 2SEQ ID NO: 483 MOR10701-VH_3-QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVAVTGAVGRST 30_JH1YYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWGDEGFDIWGQGTLVTVSSSEQ ID NO: 484 MOR10701-DIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWFQQKPGKAPKSLIYGASSLQSGVPSR VKI_L1_JK2FSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTTFGQGTKLEIK Combination 3SEQ ID NO: 485 MOR10701-VH_3-QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVAVTGAVGRST 30_JH2YYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWGDEGFDIWGRGTLVTVSSSEQ ID NO: 486 MOR10701-DIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWFQQKPGKAPKSLIYGASSLQSGVPSR VKI_L1_JK2FSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTTFGQGTKLEIK Combination 4SEQ ID NO: 487 MOR10703-VH_3-EVQLVESGGGVVRPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINSQGKSTY 20_JH5YADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYHCARWGDEGFDIWGQGTLVTVSSSEQ ID NO: 488 MOR10703-DIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPEKAPKSLIYGASSLQSGVPSRVKI_L15_JK3 FSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTTFGPGTKVDIK Combination 5SEQ ID NO: 489 MOR10703-VH_3-QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVAAINSQGKSTY 33_JH2YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDIWGRGTLVTVSSSEQ ID NO: 490 MOR10703-DIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKVPKLLIYGASSLQSGVPSRVKI_A20_JK1 FSGSGSGTDFTLTISSLQPEDVATYYCQQYSSFPTTFGQGTKVEIK Combination 6SEQ ID NO: 491 MOR10703-VH_3-QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVAAINSQGKSTY 33_JH3YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGDEGFDIWGQGTMVIVSSSEQ ID NO: 492 MOR10703-DIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKVPKLLIYGASSLQSGVPSRVKI_A20_JK2 FSGSGSGTDFTLTISSLQPEDVATYYCQQYSSFPTTFGQGTKLEIK

In one embodiment, the invention pertains to a heavy chain variableregion comprising a sequence of Xaa₁-HCDR1-Xaa₂-HCDR2-Xaa₃-HCDR3-Xaa₄where the heavy chain HCDR1, HCDR2, HCDR3 are any heavy chain CDRsselected from Tables 1 and 2. For illustrative purposes only, thesequence can be:

Xaa₁-SYAMS-Xaa₂-AINSQGKSTYYADSVKG-Xaa₃-WGDEGFDI-Xaa₄ (SEQ ID NO: 493),where,

-   -   Xaa₁ is framework region of any 30 amino acids;    -   Xaa₂ is framework region of any 14 amino acids;    -   Xaa₃ is framework region of any 32 amino acids;    -   Xaa₄ is framework region of any 11 amino acids;

In one embodiment, the invention pertains to a light chain variableregion comprising a sequence of Xaa₁-LCDR1-Xaa₂-LCDR2-Xaa₃-LCDR3-Xaa₄,where the light chain LCDR1, LCDR2, LCDR3 are any light chain CDRsselected from Tables 1 and 2. For illustrative purposes only, thesequence can be:

Xaa₁-RASQGISNWLA-Xaa₂-GASSLQS-Xaa₃-QQYSSFPTT-Xaa₄ (SEQ ID NO: 494),where,

-   -   Xaa₁ is a framework region of any 23 amino acids;    -   Xaa₂ is a framework region of any 15 amino acids;    -   Xaa₃ is a framework region of any 32 amino acids; and    -   Xaa₄ is a framework region of any 10 amino acids.

The antibodies disclosed herein can be derivatives of single chainantibodies, diabodies, domain antibodies, nanobodies, and unibodies. A“single-chain antibody” (scFv) consists of a single polypeptide chaincomprising a VL domain linked to a VH domain, wherein VL domain and VHdomain are paired to form a monovalent molecule. Single chain antibodycan be prepared according to method known in the art (see, for example,Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc.Natl. Acad. Sci. USA 85:5879-5883). A “disbud” consists of two chains,each chain comprising a heavy chain variable region connected to a lightchain variable region on the same polypeptide chain connected by a shortpeptide linker, wherein the two regions on the same chain do not pairwith each other but with complementary domains on the other chain toform a bispecific molecule. Methods of preparing diabodies are known inthe art (See, e.g., Holliger et al., (1993) Proc. Natl. Acad. Sci. USA90:6444-6448, and Poljak et al., (1994) Structure 2:1121-1123). Domainantibodies (dAbs) are small functional binding units of antibodies,corresponding to the variable regions of either the heavy or lightchains of antibodies. Domain antibodies are well expressed in bacterial,yeast, and mammalian cell systems. Further details of domain antibodiesand methods of production thereof are known in the art (see, forexample, U.S. Pat. Nos. 6,291,158; 6,582,915; 6,593,081; 6,172,197;6,696,245; European Patents 0368684 & 0616640; WO05/035572, WO04/101790,WO04/081026, WO04/058821, WO04/003019 and WO03/002609. Nanobodies arederived from the heavy chains of an antibody. A nanobody typicallycomprises a single variable domain and two constant domains (CH2 andCH3) and retains antigen-binding capacity of the original antibody.Nanobodies can be prepared by methods known in the art (See e.g., U.S.Pat. Nos. 6,765,087, 6,838,254, WO 06/079372). Unibodies consist of onelight chain and one heavy chain of a IgG4 antibody. Unibodies may bemade by the removal of the hinge region of IgG4 antibodies. Furtherdetails of unibodies and methods of preparing them may be found inWO2007/059782.

Homologous Antibodies

In yet another embodiment, the present invention provides an antibody orfragment thereof comprising amino acid sequences that are homologous tothe sequences described in Table 1, and said antibody binds to a HER3protein (e.g., human and/or cynomologus HER3), and retains the desiredfunctional properties of those antibodies described in Table 1.

For example, the invention provides an isolated monoclonal antibody (ora functional fragment thereof) comprising a heavy chain variable regionand a light chain variable region, wherein the heavy chain variableregion comprises an amino acid sequence that is at least 80%, at least90%, or at least 95% identical to an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 15, 33, 51, 69, 87, 105, 123, 141,159, 177, 195, 213, 231, 249, 267, 285, 303, 321, 339, 357, and 375; thelight chain variable region comprises an amino acid sequence that is atleast 80%, at least 90%, or at least 95% identical to an amino acidsequence selected from the group consisting of SEQ ID NOs: 14, 32, 50,68, 86, 104, 122, 140, 158, 176, 194, 212, 230, 248, 266, 284, 302, 320,338, 356, and 374; the antibody binds to HER3 (e.g., human and/orcynomologus HER3) and neutralizes the signaling activity of HER3, whichcan be measured in a phosphorylation assay or other measure of HERsignaling (e.g., phospo-HER3 assays, phospho-Akt assays, cellproliferation, and ligand blocking assays as described in the Examples).Also includes within the scope of the invention are variable heavy andlight chain parental nucleotide sequences; and full length heavy andlight chain sequences optimized for expression in a mammalian cell.Other antibodies of the invention include amino acids or nucleic acidsthat have been mutated, yet have at least 60, 70, 80, 90, 95, or 98%percent identity to the sequences described above. In some embodiments,it include mutant amino acid sequences wherein no more than 1, 2, 3, 4or 5 amino acids have been mutated by amino acid deletion, insertion orsubstitution in the variable regions when compared with the variableregions depicted in the sequence described above.

In other embodiments, the VH and/or VL amino acid sequences may be 50%,60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequencesset forth in Table 1. In other embodiments, the VH and/or VL amino acidsequences may be identical except an amino acid substitution in no morethan 1, 2, 3, 4 or 5 amino acid position. An antibody having VH and VLregions having high (i. e., 80% or greater) identity to the VH and VLregions of the antibodies described in Table 1 can be obtained bymutagenesis (e.g., site-directed or PCR-mediated mutagenesis), followedby testing of the encoded altered antibody for retained function usingthe functional assays described herein.

In other embodiments, the variable regions of heavy chain and/or lightchain nucleotide sequences may be 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%or 99% identical to the sequences set forth above.

As used herein, “percent identity” between the two sequences is afunction of the number of identical positions shared by the sequences(i.e., % identity equals number of identical positions/total number ofpositions×100), taking into account the number of gaps, and the lengthof each gap, which needs to be introduced for optimal alignment of thetwo sequences. The comparison of sequences and determination of percentidentity between two sequences can be accomplished using a mathematicalalgorithm, as described in the non-limiting examples below.

Additionally or alternatively, the protein sequences of the presentinvention can further be used as a “query sequence” to perform a searchagainst public databases to, for example, identifies related sequences.For example, such searches can be performed using the BLAST program(version 2.0) of Altschul et al., (1990) J. Mol. Biol. 215:403-10.

Antibodies with Conservative Modifications

In certain embodiments, an antibody of the invention has a heavy chainvariable region comprising CDR1, CDR2, and CDR3 sequences and a lightchain variable region comprising CDR1, CDR2, and CDR3 sequences, whereinone or more of these CDR sequences have specified amino acid sequencesbased on the antibodies described herein or conservative modificationsthereof, and wherein the antibodies retain the desired functionalproperties of the HER3-binding antibodies of the invention.

Accordingly, the invention provides an isolated HER3 monoclonalantibody, or a fragment thereof, consisting of a heavy chain variableregion comprising CDR1, CDR2, and CDR3 sequences and a light chainvariable region comprising CDR1, CDR2, and CDR3 sequences, wherein: theheavy chain variable region CDR1 amino acid sequences are selected fromthe group consisting of SEQ ID NOs: 2, 8, 20, 26, 38, 44, 56, 62, 74,80, 92, 98, 110, 116, 128, 134, 146, 152, 164, 170, 182, 188, 200, 206,218, 224, 236, 242, 254, 260, 272, 278, 290, 296, 308, 314, 326, 332,344, 350, 362, and 368, and conservative modifications thereof; theheavy chain variable region CDR2 amino acid sequences are selected fromthe group consisting of SEQ ID NOs: 3, 9, 21, 27, 39, 45, 57, 63, 75,81, 93, 99, 111, 117, 129, 135, 147, 153, 165, 171, 183, 189, 201, 207,219, 225, 237, 243, 255, 261, 273, 279, 291, 297, 309, 315, 327, 333,345, 351, 363, and 369 and conservative modifications thereof, the heavychain variable region CDR3 amino acid sequences are selected from thegroup consisting of SEQ ID NOs: 4, 10, 22, 28, 40, 46, 58, 64, 76, 82,94, 100, 112, 118, 130, 136, 148, 154, 166, 172, 184, 190, 202, 208,220, 226, 238, 244, 256, 262, 274, 280, 292, 298, 310, 316, 328, 334,346, 352, 364, and 370 and conservative modifications thereof, the lightchain variable regions CDR1 amino acid sequences are selected from thegroup consisting of SEQ ID NOs: 5, 11, 23, 29, 41, 47, 59, 65, 77, 83,95, 101, 113, 119, 131, 137, 149, 155, 167, 173, 185, 191, 203, 209,221, 227, 239, 245, 257, 263, 275, 281, 293, 299, 311, 317, 329, 335,347, 353, 365, and 371 and conservative modifications thereof, the lightchain variable regions CDR2 amino acid sequences are selected from thegroup consisting of SEQ ID NOs: 6, 12, 24, 30, 42, 48, 60, 66, 78, 84,96, 102, 114, 120, 132, 138, 150, 156, 168, 174, 186, 192, 204, 210,222, 228, 240, 246, 258, 264, 276, 282, 294, 300, 312, 318, 330, 336,348, 354, 366, and 372, and conservative modifications thereof, thelight chain variable regions of CDR3 amino acid sequences are selectedfrom the group consisting of SEQ ID NOs: 7, 13, 25, 31, 43, 49, 61, 67,79, 85, 97, 103, 115, 121, 133, 139, 151, 157, 169, 175, 187, 193, 205,211, 223, 229, 241, 247, 259, 265, 277, 283, 295, 301, 313, 319, 331,337, 349, 355, 367, and 373, and conservative modifications thereof, theantibody or fragment thereof specifically binds to HER3, and neutralizesHER3 activity by inhibiting a HER signaling pathway, which can bemeasured in a phosphorylation assay or other measure of HER signaling(e.g., phospo-HER3 assays, phospho-Akt assays, cell proliferation, andligand blocking assays as described in the Examples).

Antibodies that Bind to the Same Epitope

The present invention provides antibodies that interacts with (e.g., bybinding, steric hindrance, stabilizing/destabilizing, spatialdistribution) the same epitope as do the HER3-binding antibodiesdescribed in Table 1 and FIG. 7. Additional antibodies can therefore beidentified based on their ability to cross-compete (e.g., tocompetitively inhibit the binding of, in a statistically significantmanner) with other antibodies of the invention in HER3 binding assays.The ability of a test antibody to inhibit the binding of antibodies ofthe present invention to a HER3 protein (e.g., human and/or cynomologusHER3) demonstrates that the test antibody can compete with that antibodyfor binding to HER3; such an antibody may, according to non-limitingtheory, bind to the same or a related (e.g., a structurally similar orspatially proximal) epitope on the HER3 protein as the antibody withwhich it competes. In a certain embodiment, the antibody that binds tothe same epitope on HER3 as the antibodies of the present invention is ahuman monoclonal antibody. Such human monoclonal antibodies can beprepared and isolated as described herein.

In one embodiment, the antibody or fragments thereof binds to bothdomain 2 and domain 4 of HER3 to hold the HER3 in an inactiveconformation which prevents exposure of an dimerization loop presentwithin domain 2. This prevents heterodimerization with other familymembers, such as HER1, HER2, and HER4. The antibodies of fragmentsthereof inhibit both ligand dependent and ligand-independent HER3 signaltransduction.

In another embodiment, the antibody or fragment thereof binds to bothdomain 2 and domain 4 of HER3 and without blocking the concurrentbinding of a HER3 ligand such as neuregulin. While not required toprovide a theory, it is feasible that the antibody or fragment thereofbinding to both domain 2 and domain 4 of HER3, holds HER3 in an inactiveconformation without blocking the ligand binding site on HER3. Thus aHER3 ligand (e.g., neuregulin) is able to bind to HER3 at the same timeas the antibody or fragment thereof.

The antibodies of the invention or fragments thereof inhibit both liganddependent and independent activation of HER3 without preventing ligandbinding. This is considered advantageous for the following reasons:

(i) The therapeutic antibody would have clinical utility in a broadspectrum of tumors than an antibody which targeted a single mechanism ofHER3 activation (i.e. ligand dependent or ligand independent) sincedistinct tumor types are driven by each mechanism.

(ii) The therapeutic antibody would be efficacious in tumor types whereboth mechanisms of HER3 activation are simultaneously involved. Anantibody targeting a single mechanism of HER3 activation (i.e. liganddependent or ligand independent) would display little or no efficacy inthese tumor types

(iii) The efficacy of an antibody which inhibits ligand dependentactivation of HER3 without preventing ligand binding would be lesslikely to be adversely affected by increasing concentrations of ligand.This would translate to either increased efficacy in a tumor type drivenby very high concentrations of HER3 ligand or a reduced drug resistanceliability where resistance is mediated by up-regulation of HER3 ligands.

(iv) An antibody which inhibits HER3 activation by stabilizing theinactive form would be less prone to drug resistance driven byalternative mechanisms of HER3 activation.

Consequently, the antibodies of the invention may be used to treatconditions where existing therapeutic antibodies are clinicallyineffective.

Engineered and Modified Antibodies

An antibody of the invention further can be prepared using an antibodyhaving one or more of the VH and/or VL sequences shown herein asstarting material to engineer a modified antibody, which modifiedantibody may have altered properties from the starting antibody. Anantibody can be engineered by modifying one or more residues within oneor both variable regions (i. e., VH and/or VL), for example within oneor more CDR regions and/or within one or more framework regions.Additionally or alternatively, an antibody can be engineered bymodifying residues within the constant region(s), for example to alterthe effector function(s) of the antibody.

One type of variable region engineering that can be performed is CDRgrafting. Antibodies interact with target antigens predominantly throughamino acid residues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann et al., (1998) Nature332:323-327; Jones et al., (1986) Nature 321:522-525; Queen et al.,(1989) Proc. Natl. Acad., U.S.A. 86:10029-10033; U.S. Pat. No. 5,225,539to Winter, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and6,180,370 to Queen et al.)

Accordingly, another embodiment of the invention pertains to an isolatedHER3 binding monoclonal antibody, or fragment thereof, comprising aheavy chain variable region comprising CDR1 sequences having an aminoacid sequence selected from the group consisting of SEQ ID NOs: 2, 8,20, 26, 38, 44, 56, 62, 74, 80, 92, 98, 110, 116, 128, 134, 146, 152,164, 170, 182, 188, 200, 206, 218, 224, 236, 242, 254, 260, 272, 278,290, 296, 308, 314, 326, 332, 344, 350, 362, and 368; CDR2 sequenceshaving an amino acid sequence selected from the group consisting of SEQID NOs: 3, 9, 21, 27, 39, 45, 57, 63, 75, 81, 93, 99, 111, 117, 129,135, 147, 153, 165, 171, 183, 189, 201, 207, 219, 225, 237, 243, 255,261, 273, 279, 291, 297, 309, 315, 327, 333, 345, 351, 363, and 369;CDR3 sequences having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 4, 10, 22, 28, 40, 46, 58, 64, 76, 82, 94,100, 112, 118, 130, 136, 148, 154, 166, 172, 184, 190, 202, 208, 220,226, 238, 244, 256, 262, 274, 280, 292, 298, 310, 316, 328, 334, 346,352, 364, and 370, respectively; and a light chain variable regionhaving CDR1 sequences having an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 5, 11, 23, 29, 41, 47, 59, 65, 77, 83,95, 101, 113, 119, 131, 137, 149, 155, 167, 173, 185, 191, 203, 209,221, 227, 239, 245, 257, 263, 275, 281, 293, 299, 311, 317, 329, 335,347, 353, 365, and 371; CDR2 sequences having an amino acid sequenceselected from the group consisting of SEQ ID NOs: 6, 12, 24, 30, 42, 48,60, 66, 78, 84, 96, 102, 114, 120, 132, 138, 150, 156, 168, 174, 186,192, 204, 210, 222, 228, 240, 246, 258, 264, 276, 282, 294, 300, 312,318, 330, 336, 348, 354, 366, and 372; and CDR3 sequences consisting ofan amino acid sequence selected from the group consisting of SEQ ID NOs:7, 13, 25, 31, 43, 49, 61, 67, 79, 85, 97, 103, 115, 121, 135, 139, 151,157, 169, 175, 187, 193, 205, 211, 223, 229, 241, 247, 259, 265, 277,283, 295, 301, 313, 319, 331, 337, 349, 355, 367, and 373, respectively.Thus, such antibodies contain the VH and VL CDR sequences of monoclonalantibodies, yet may contain different framework sequences from theseantibodies. Such framework sequences can be obtained from public DNAdatabases or published references that include germline antibody genesequences. For example, germline DNA sequences for human heavy and lightchain variable region genes can be found in the “Vase” human germlinesequence database (available on the Internet atwww.mrc-cpe.cam.ac.uk/vbase), as well as in Kabat et al., (1991)Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242;Chothia et al., (1987) J. Mol. Biol. 196:901-917; Chothia et al., (1989)Nature 342:877-883; and Al-Lazikani et al., (1997) J. Mol. Biol.273:927-948; Tomlinson et al., (1992) J. fol. Biol. 227:776-798; and Coxet al., (1994) Eur. J Immunol. 24:827-836; the contents of each of whichare expressly incorporated herein by reference.

An example of framework sequences for use in the antibodies of theinvention are those that are structurally similar to the frameworksequences used by selected antibodies of the invention, e.g., consensussequences and/or framework sequences used by monoclonal antibodies ofthe invention. The VH CDR1, 2 and 3 sequences, and the VL CDR1, 2 and 3sequences, can be grafted onto framework regions that have the identicalsequence as that found in the germline immunoglobulin gene from whichthe framework sequence derive, or the CDR sequences can be grafted ontoframework regions that contain one or more mutations as compared to thegermline sequences. For example, it has been found that in certaininstances it is beneficial to mutate residues within the frameworkregions to maintain or enhance the antigen binding ability of theantibody (see e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and6,180,370 to Queen et al).

Another type of variable region modification is to mutate amino acidresidues within the VH and/or VL CDR1, CDR2 and/or CDR3 regions tothereby improve one or more binding properties (e.g., affinity) of theantibody of interest, known as “affinity maturation.” Site-directedmutagenesis or PCR-mediated mutagenesis can be performed to introducethe mutation(s) and the effect on antibody binding, or other functionalproperty of interest, can be evaluated in in vitro or in vivo assays asdescribed herein and provided in the Examples. Conservativemodifications (as discussed above) can be introduced. The mutations maybe amino acid substitutions, additions or deletions. Moreover, typicallyno more than one, two, three, four or five residues within a CDR regionare altered.

Accordingly, in another embodiment, the invention provides isolated HER3binding monoclonal antibodies, or fragment thereof, consisting of aheavy chain variable region having: a VH CDR1 region consisting of anamino acid sequence selected from the group having SEQ ID NOs: 2, 8, 20,26, 38, 44, 56, 62, 74, 80, 92, 98, 110, 116, 128, 134, 146, 152, 164,170, 182, 188, 200, 206, 218, 224, 236, 242, 254, 260, 272, 278, 290,296, 308, 314, 326, 332, 344, 350, 362, and 368 or an amino acidsequence having one, two, three, four or five amino acid substitutions,deletions or additions as compared to SEQ ID NOs: 2, 8, 20, 26, 38, 44,56, 62, 74, 80, 92, 98, 110, 116, 128, 134, 146, 152, 164, 170, 182,188, 200, 206, 218, 224, 236, 242, 254, 260, 272, 278, 290, 296, 308,314, 326, 332, 344, 350, 362, and 368; a VH CDR2 region having an aminoacid sequence selected from the group consisting of SEQ ID NOs: 3, 9,21, 27, 39, 45, 57, 63, 75, 81, 93, 99, 111, 117, 129, 135, 147, 153,165, 171, 183, 189, 201, 207, 219, 225, 237, 243, 255, 261, 273, 279,291, 297, 309, 315, 327, 333, 345, 351, 363, and 369 or an amino acidsequence having one, two, three, four or five amino acid substitutions,deletions or additions as compared to SEQ ID NOs: 3, 9, 21, 27, 39, 45,57, 63, 75, 81, 93, 99, 111, 117, 129, 135, 147, 153, 165, 171, 183,189, 201, 207, 219, 225, 237, 243, 255, 261, 273, 279, 291, 297, 309,315, 327, 333, 345, 351, 363, and 369; a VH CDR3 region having an aminoacid sequence selected from the group consisting of SEQ ID NOs: 4, 10,22, 28, 40, 46, 58, 64, 76, 82, 94, 100, 112, 118, 130, 136, 148, 154,166, 172, 184, 190, 202, 208, 220, 226, 238, 244, 256, 262, 274, 280,292, 298, 310, 316, 328, 334, 346, 352, 364, and 370, or an amino acidsequence having one, two, three, four or five amino acid substitutions,deletions or additions as compared to SEQ ID NOs: 4, 10, 22, 28, 40, 46,58, 64, 76, 82, 94, 100, 112, 118, 130, 136, 148, 154, 166, 172, 184,190, 202, 208, 220, 226, 238, 244, 256, 262, 274, 280, 292, 298, 310,316, 328, 334, 346, 352, 364, and 370; a VL CDR1 region having an aminoacid sequence selected from the group consisting of SEQ ID NOs: 5, 11,23, 29, 41, 47, 59, 65, 77, 83, 95, 101, 113, 119, 131, 137, 149, 155,167, 173, 185, 191, 203, 209, 221, 227, 239, 245, 257, 263, 275, 281,293, 299, 311, 317, 329, 335, 347, 353, 365, and 371, or an amino acidsequence having one, two, three, four or five amino acid substitutions,deletions or additions as compared to SEQ ID NOs: 5, 11, 23, 29, 41, 47,59, 65, 77, 83, 95, 101, 113, 119, 131, 137, 149, 155, 167, 173, 185,191, 203, 209, 221, 227, 239, 245, 257, 263, 275, 281, 293, 299, 311,317, 329, 335, 347, 353, 365, and 371; a VL CDR2 region having an aminoacid sequence selected from the group consisting of SEQ ID NOs: 6, 12,24, 30, 42, 48, 60, 66, 78, 84, 96, 102, 114, 120, 132, 138, 150, 156,168, 174, 186, 192, 204, 210, 222, 228, 240, 246, 258, 264, 276, 282,294, 300, 312, 318, 330, 336, 348, 354, 366, and 372, or an amino acidsequence having one, two, three, four or five amino acid substitutions,deletions or additions as compared to SEQ ID NOs: 6, 12, 24, 30, 42, 48,60, 66, 78, 84, 96, 102, 114, 120, 132, 138, 150, 156, 168, 174, 186,192, 204, 210, 222, 228, 240, 246, 258, 264, 276, 282, 294, 300, 312,318, 330, 336, 348, 354, 366, and 372; and a VL CDR3 region having anamino acid sequence selected from the group consisting of SEQ ID NOs: 7,13, 25, 31, 43, 49, 61, 67, 79, 85, 97, 103, 115, 121, 135, 139, 139,151, 157, 169, 175, 187, 193, 205, 211, 223, 229, 241, 247, 259, 265,277, 283, 295, 301, 313, 319, 331, 337, 349, 355, 367, and 373, or anamino acid sequence having one, two, three, four or five amino acidsubstitutions, deletions or additions as compared to SEQ ID NOs: 7, 13,25, 31, 43, 49, 61, 67, 79, 85, 97, 103, 115, 121, 135, 139, 139, 151,157, 169, 175, 187, 193, 205, 211, 223, 229, 241, 247, 259, 265, 277,283, 295, 301, 313, 319, 331, 337, 349, 355, 367, and 373.

Grafting Antibody Fragments Into Alternative Frameworks or Scaffolds Awide variety of antibody/immunoglobulin frameworks or scaffolds can beemployed so long as the resulting polypeptide includes at least onebinding region which specifically binds to HER3. Such frameworks orscaffolds include the 5 main idiotypes of human immunoglobulins, orfragments thereof, and include immunoglobulins of other animal species,preferably having humanized aspects. Novel frameworks, scaffolds andfragments continue to be discovered and developed by those skilled inthe art.

In one aspect, the invention pertains to generating non-immunoglobulinbased antibodies using non-immunoglobulin scaffolds onto which CDRs ofthe invention can be grafted. Known or future non-immunoglobulinframeworks and scaffolds may be employed, as long as they comprise abinding region specific for the target HER3 protein (e.g., human and/orcynomologus HER3). Known non-immunoglobulin frameworks or scaffoldsinclude, but are not limited to, fibronectin (Compound Therapeutics,Inc., Waltham, Mass.), ankyrin (Molecular Partners AG, Zurich,Switzerland), domain antibodies (Domantis, Ltd., Cambridge, Mass., andAblynx nv, Zwijnaarde, Belgium), lipocalin (Pieris Proteolab AG,Freising, Germany), small modular immuno-pharmaceuticals (TrubionPharmaceuticals Inc., Seattle, Wash.), maxybodies (Avidia, Inc.,Mountain View, Calif.), Protein A (Affibody AG, Sweden), and affilin(gamma-crystallin or ubiquitin) (Scil Proteins GmbH, Halle, Germany).The fibronectin scaffolds are based on fibronectin type III domain(e.g., the tenth module of the fibronectin type III (¹⁰Fn3 domain)). Thefibronectin type III domain has 7 or 8 beta strands which aredistributed between two beta sheets, which themselves pack against eachother to form the core of the protein, and further containing loops(analogous to CDRs) which connect the beta strands to each other and aresolvent exposed. There are at least three such loops at each edge of thebeta sheet sandwich, where the edge is the boundary of the proteinperpendicular to the direction of the beta strands (see U.S. Pat. No.6,818,418). These fibronectin-based scaffolds are not an immunoglobulin,although the overall fold is closely related to that of the smallestfunctional antibody fragment, the variable region of the heavy chain,which comprises the entire antigen recognition unit in camel and llamaIgG. Because of this structure, the non-immunoglobulin antibody mimicsantigen binding properties that are similar in nature and affinity tothose of antibodies. These scaffolds can be used in a loop randomizationand shuffling strategy in vitro that is similar to the process ofaffinity maturation of antibodies in vivo. These fibronectin-basedmolecules can be used as scaffolds where the loop regions of themolecule can be replaced with CDRs of the invention using standardcloning techniques.

The ankyrin technology is based on using proteins with ankyrin derivedrepeat modules as scaffolds for bearing variable regions which can beused for binding to different targets. The ankyrin repeat module is a 33amino acid polypeptide consisting of two anti-parallel α-helices and aβ-turn. Binding of the variable regions is mostly optimized by usingribosome display.

Avimers are derived from natural A-domain containing protein such asHER3. These domains are used by nature for protein-protein interactionsand in human over 250 proteins are structurally based on A-domains.Avimers consist of a number of different “A-domain” monomers (2-10)linked via amino acid linkers. Avimers can be created that can bind tothe target antigen using the methodology described in, for example, U.S.Patent Application Publication Nos. 20040175756; 20050053973;20050048512; and 20060008844.

Affibody affinity ligands are small, simple proteins composed of athree-helix bundle based on the scaffold of one of the IgG-bindingdomains of Protein A. Protein A is a surface protein from the bacteriumStaphylococcus aureus. This scaffold domain consists of 58 amino acids,13 of which are randomized to generate affibody libraries with a largenumber of ligand variants (See e.g., U.S. Pat. No. 5,831,012). Affibodymolecules mimic antibodies, they have a molecular weight of 6 kDa,compared to the molecular weight of antibodies, which is 150 kDa. Inspite of its small size, the binding site of affibody molecules issimilar to that of an antibody.

Anticalins are products developed by the company Pieris ProteoLab AG.They are derived from lipocalins, a widespread group of small and robustproteins that are usually involved in the physiological transport orstorage of chemically sensitive or insoluble compounds. Several naturallipocalins occur in human tissues or body liquids. The proteinarchitecture is reminiscent of immunoglobulins, with hypervariable loopson top of a rigid framework. However, in contrast with antibodies ortheir recombinant fragments, lipocalins are composed of a singlepolypeptide chain with 160 to 180 amino acid residues, being justmarginally bigger than a single immunoglobulin domain. The set of fourloops, which makes up the binding pocket, shows pronounced structuralplasticity and tolerates a variety of side chains. The binding site canthus be reshaped in a proprietary process in order to recognizeprescribed target molecules of different shape with high affinity andspecificity. One protein of lipocalin family, the bilin-binding protein(BBP) of Pieris Brassicae has been used to develop anticalins bymutagenizing the set of four loops. One example of a patent applicationdescribing anticalins is in PCT Publication No. WO 199916873.

Affilin molecules are small non-immunoglobulin proteins which aredesigned for specific affinities towards proteins and small molecules.New affilin molecules can be very quickly selected from two libraries,each of which is based on a different human derived scaffold protein.Affilin molecules do not show any structural homology to immunoglobulinproteins. Currently, two affilin scaffolds are employed, one of which isgamma crystalline, a human structural eye lens protein and the other is“ubiquitin” superfamily proteins. Both human scaffolds are very small,show high temperature stability and are almost resistant to pH changesand denaturing agents. This high stability is mainly due to the expandedbeta sheet structure of the proteins. Examples of gamma crystallinederived proteins are described in WO200104144 and examples of“ubiquitin-like” proteins are described in WO2004106368.

Protein epitope mimetics (PEM) are medium-sized, cyclic, peptide-likemolecules (MW 1-2 kDa) mimicking beta-hairpin secondary structures ofproteins, the major secondary structure involved in protein-proteininteractions.

In some embodiments, the Fabs are converted to silent IgG1 format bychanging the Fc region. For example, antibodies in Table 1 can beconverted to IgG format.

Human or Humanized Antibodies

The present invention provides fully human antibodies that specificallybind to a HER3 protein (e.g., human and/or cynomologus/mouse/rat HER3).Compared to the chimeric or humanized antibodies, the human HER3-bindingantibodies of the invention have further reduced antigenicity whenadministered to human subjects.

The human HER3-binding antibodies can be generated using methods thatare known in the art. For example, the humaneering technology used toconverting non-human antibodies into engineered human antibodies. U.S.Patent Publication No. 20050008625 describes an in vivo method forreplacing a nonhuman antibody variable region with a human variableregion in an antibody while maintaining the same or providing betterbinding characteristics relative to that of the nonhuman antibody. Themethod relies on epitope guided replacement of variable regions of anon-human reference antibody with a fully human antibody. The resultinghuman antibody is generally unrelated structurally to the referencenonhuman antibody, but binds to the same epitope on the same antigen asthe reference antibody. Briefly, the serial epitope-guidedcomplementarity replacement approach is enabled by setting up acompetition in cells between a “competitor” and a library of diversehybrids of the reference antibody (“test antibodies”) for binding tolimiting amounts of antigen in the presence of a reporter system whichresponds to the binding of test antibody to antigen. The competitor canbe the reference antibody or derivative thereof such as a single-chainFv fragment. The competitor can also be a natural or artificial ligandof the antigen which binds to the same epitope as the referenceantibody. The only requirements of the competitor are that it binds tothe same epitope as the reference antibody, and that it competes withthe reference antibody for antigen binding. The test antibodies have oneantigen-binding V-region in common from the nonhuman reference antibody,and the other V-region selected at random from a diverse source such asa repertoire library of human antibodies. The common V-region from thereference antibody serves as a guide, positioning the test antibodies onthe same epitope on the antigen, and in the same orientation, so thatselection is biased toward the highest antigen-binding fidelity to thereference antibody.

Many types of reporter system can be used to detect desired interactionsbetween test antibodies and antigen. For example, complementing reporterfragments may be linked to antigen and test antibody, respectively, sothat reporter activation by fragment complementation only occurs whenthe test antibody binds to the antigen. When the test antibody- andantigen-reporter fragment fusions are co-expressed with a competitor,reporter activation becomes dependent on the ability of the testantibody to compete with the competitor, which is proportional to theaffinity of the test antibody for the antigen. Other reporter systemsthat can be used include the reactivator of an auto-inhibited reporterreactivation system (RAIR) as disclosed in U.S. patent application Ser.No. 10/208,730 (Publication No. 20030198971), or competitive activationsystem disclosed in U.S. patent application Ser. No. 10/076,845(Publication No. 20030157579).

With the serial epitope-guided complementarity replacement system,selection is made to identify cells expresses a single test antibodyalong with the competitor, antigen, and reporter components. In thesecells, each test antibody competes one-on-one with the competitor forbinding to a limiting amount of antigen. Activity of the reporter isproportional to the amount of antigen bound to the test antibody, whichin turn is proportional to the affinity of the test antibody for theantigen and the stability of the test antibody. Test antibodies areinitially selected on the basis of their activity relative to that ofthe reference antibody when expressed as the test antibody. The resultof the first round of selection is a set of “hybrid” antibodies, each ofwhich is comprised of the same non-human V-region from the referenceantibody and a human V-region from the library, and each of which bindsto the same epitope on the antigen as the reference antibody. One ofmore of the hybrid antibodies selected in the first round will have anaffinity for the antigen comparable to or higher than that of thereference antibody.

In the second V-region replacement step, the human V-regions selected inthe first step are used as guide for the selection of human replacementsfor the remaining non-human reference antibody V-region with a diverselibrary of cognate human V-regions. The hybrid antibodies selected inthe first round may also be used as competitors for the second round ofselection. The result of the second round of selection is a set of fullyhuman antibodies which differ structurally from the reference antibody,but which compete with the reference antibody for binding to the sameantigen. Some of the selected human antibodies bind to the same epitopeon the same antigen as the reference antibody. Among these selectedhuman antibodies, one or more binds to the same epitope with an affinitywhich is comparable to or higher than that of the reference antibody.

Using one of the mouse or chimeric HER3-binding antibodies describedabove as the reference antibody, this method can be readily employed togenerate human antibodies that bind to human HER3 with the same bindingspecificity and the same or better binding affinity. In addition, suchhuman HER3-binding antibodies can also be commercially obtained fromcompanies which customarily produce human antibodies, e.g., KaloBios,Inc. (Mountain View, Calif.).

Camelid Antibodies

Antibody proteins obtained from members of the camel and dromedary(Camelus bactrianus and Calelus dromaderius) family including new worldmembers such as llama species (Lama paccos, Lama glama and Lama vicugna)have been characterized with respect to size, structural complexity andantigenicity for human subjects. Certain IgG antibodies from this familyof mammals as found in nature lack light chains, and are thusstructurally distinct from the typical four chain quaternary structurehaving two heavy and two light chains, for antibodies from otheranimals. See PCT/EP93/02214 (WO 94/04678 published 3 Mar. 1994).

A region of the camelid antibody which is the small single variabledomain identified as VHH can be obtained by genetic engineering to yielda small protein having high affinity for a target, resulting in a lowmolecular weight antibody-derived protein known as a “camelid nanobody”.See U.S. Pat. No. 5,759,808 issued Jun. 2, 1998; see also Stijlemans etal., (2004) J Biol Chem 279:1256-1261; Dumoulin et al., (2003) Nature424:783-788; Pleschberger et al., (2003) Bioconjugate Chem 14:440-448;Cortez-Retamozo et al., (2002) Int J Cancer 89:456-62; and Lauwereys etal., (1998) EMBO J 17:3512-3520. Engineered libraries of camelidantibodies and antibody fragments are commercially available, forexample, from Ablynx, Ghent, Belgium. (e.g., US20060115470; Domantis(US20070065440, US20090148434). As with other antibodies of non-humanorigin, an amino acid sequence of a camelid antibody can be alteredrecombinantly to obtain a sequence that more closely resembles a humansequence, i.e., the nanobody can be “humanized”. Thus the natural lowantigenicity of camelid antibodies to humans can be further reduced.

The camelid nanobody has a molecular weight approximately one-tenth thatof a human IgG molecule, and the protein has a physical diameter of onlya few nanometers. One consequence of the small size is the ability ofcamelid nanobodies to bind to antigenic sites that are functionallyinvisible to larger antibody proteins, i.e., camelid nanobodies areuseful as reagents detect antigens that are otherwise cryptic usingclassical immunological techniques, and as possible therapeutic agents.Thus yet another consequence of small size is that a camelid nanobodycan inhibit as a result of binding to a specific site in a groove ornarrow cleft of a target protein, and hence can serve in a capacity thatmore closely resembles the function of a classical low molecular weightdrug than that of a classical antibody.

The low molecular weight and compact size further result in camelidnanobodies being extremely thermostable, stable to extreme pH and toproteolytic digestion, and poorly antigenic. Another consequence is thatcamelid nanobodies readily move from the circulatory system intotissues, and even cross the blood-brain barrier and can treat disordersthat affect nervous tissue. Nanobodies can further facilitated drugtransport across the blood brain barrier. See U.S. patent application20040161738 published Aug. 19, 2004. These features combined with thelow antigenicity to humans indicate great therapeutic potential.Further, these molecules can be fully expressed in prokaryotic cellssuch as E. coli and are expressed as fusion proteins with bacteriophageand are functional.

Accordingly, a feature of the present invention is a camelid antibody ornanobody having high affinity for HER3. In certain embodiments herein,the camelid antibody or nanobody is naturally produced in the camelidanimal, i.e., is produced by the camelid following immunization withHER3 or a peptide fragment thereof, using techniques described hereinfor other antibodies. Alternatively, the HER3-binding camelid nanobodyis engineered, i.e., produced by selection for example from a library ofphage displaying appropriately mutagenized camelid nanobody proteinsusing panning procedures with HER3 as a target as described in theexamples herein. Engineered nanobodies can further be customized bygenetic engineering to have a half life in a recipient subject of from45 minutes to two weeks. In a specific embodiment, the camelid antibodyor nanobody is obtained by grafting the CDRs sequences of the heavy orlight chain of the human antibodies of the invention into nanobody orsingle domain antibody framework sequences, as described for example inPCT/EP93/02214. In one embodiment, the camelid antibody or nanobodybinds to at least one of the following HER3 residues: Asn266, Lys267,Leu268, Thr269, Gln271, Glu273, Pro274, Asn275, Pro276, His277, Asn315,Asp571, Pro583, His584, Ala596, Lys597 (residues of SEQ ID NO: 1). Inone embodiment, the camelid antibody or nanobody binds to at least oneof the following HER3 residues: Tyr265, Lys267, Leu268, Phe270, Gly582,Pro583, Lys597, Ile600, Lys602, Glu609, Arg611, Pro612, Cys613, His614,Glu615 (residues of SEQ ID NO: 1).

Bispecific Molecules and Multivalent Antibodies

In another aspect, the present invention features biparatopic,bispecific or multispecific molecules comprising an HER3-bindingantibody, or a fragment thereof, of the invention. An antibody of theinvention, or fragments thereof, can be derivatized or linked to anotherfunctional molecule, e.g., another peptide or protein (e.g., anotherantibody or ligand for a receptor) to generate a bispecific moleculethat binds to at least two different binding sites or target molecules.The antibody of the invention may in fact be derivatized or linked tomore than one other functional molecule to generate biparatopic ormulti-specific molecules that bind to more than two different bindingsites and/or target molecules; such biparatopic or multi-specificmolecules. To create a bispecific molecule of the invention, an antibodyof the invention can be functionally linked (e.g., by chemical coupling,genetic fusion, non-covalent association or otherwise) to one or moreother binding molecules, such as another antibody, antibody fragment,peptide or binding mimetic, such that a bispecific molecule results.

Further clinical benefits may be provided by the binding of two or moreantigens within one antibody (Coloma et al., (1997); Merchant et al.,(1998); Alt et al., (1999); Zuo et al., (2000); Lu et al., (2004); Lu etal., (2005); Marvin et al., (2005); Marvin et al., (2006); Shen et al.,(2007); Wu et al., (2007); Dimasi et al., (2009); Michaelson et al.,(2009)). (Morrison et al., (1997) Nature Biotech. 15:159-163; Alt et al.(1999) FEBS Letters 454:90-94; Zuo et al., (2000) Protein Engineering13:361-367; Lu et al., (2004) JBC 279:2856-2865; Lu et al., (2005) JBC280:19665-19672; Marvin et al., (2005) Acta Pharmacologica Sinica26:649-658; Marvin et al., (2006) Curr Opin Drug Disc Develop 9:184-193;Shen et al., (2007) J Immun Methods 218:65-74; Wu et al., (2007) NatBiotechnol. 11:1290-1297; Dimasi et al., (2009) J Mol Biol. 393:672-692;and Michaelson et al., (2009) mAbs 1:128-141.

The bispecific molecules of the present invention can be prepared byconjugating the constituent binding specificities, using methods knownin the art. For example, each binding specificity of the bispecificmolecule can be generated separately and then conjugated to one another.When the binding specificities are proteins or peptides, a variety ofcoupling or cross-linking agents can be used for covalent conjugation.Examples of cross-linking agents include protein A, carbodiimide,N-succinimidyl-S-acetyl-thioacetate (SATA),5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide(oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), andsulfosuccinimidyl 4-(N-maleimidomethyl) cyclohaxane-1-carboxylate(sulfo-SMCC) (see e.g., Karpovsky et al., (1984) J. Exp. Med. 160:1686;Liu et al., (1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methodsinclude those described in Paulus (1985) Behring Ins. Mitt. No.78:118-132; Brennan et al., (1985) Science 229:81-83), and Glennie etal., (1987) J. Immunol. 139: 2367-2375). Conjugating agents are SATA andsulfo-SMCC, both available from Pierce Chemical Co. (Rockford, Ill.).

When the binding specificities are antibodies, they can be conjugated bysulfhydryl bonding of the C-terminus hinge regions of the two heavychains. In a particularly embodiment, the hinge region is modified tocontain an odd number of sulfhydryl residues, for example one, prior toconjugation.

Alternatively, both binding specificities can be encoded in the samevector and expressed and assembled in the same host cell. This method isparticularly useful where the bispecific molecule is a mAb x mAb, mAb xFab, Fab x F(ab′)₂ or ligand x Fab fusion protein. A bispecific moleculeof the invention can be a single chain molecule comprising one singlechain antibody and a binding determinant, or a single chain bispecificmolecule comprising two binding determinants. Bispecific molecules maycomprise at least two single chain molecules. Methods for preparingbispecific molecules are described for example in U.S. Pat. Nos.5,260,203; 5,455,030; 4,881,175; 5,132,405; 5,091,513; 5,476,786;5,013,653; 5,258,498; and 5,482,858.

Binding of the bispecific molecules to their specific targets can beconfirmed by, for example, enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (REA), FACS analysis, bioassay (e.g., growthinhibition), or Western Blot assay. Each of these assays generallydetects the presence of protein-antibody complexes of particularinterest by employing a labeled reagent (e.g., an antibody) specific forthe complex of interest.

In another aspect, the present invention provides multivalent compoundscomprising at least two identical or different fragments of theantibodies of the invention binding to HER3. The antibody fragments canbe linked together via protein fusion or covalent or non covalentlinkage. Tetravalent compounds can be obtained for example bycross-linking antibodies of the antibodies of the invention with anantibody that binds to the constant regions of the antibodies of theinvention, for example the Fc or hinge region. Trimerizing domain aredescribed for example in Borean patent EP 1012280B1. Pentamerizingmodules are described for example in PCT/EP97/05897.

In one embodiment, a biparatopic/bispecific binds to amino acid residueswithin domain 2 and domain 4 of HER3.

In another embodiment, the invention pertains to dual functionantibodies in which a single monoclonal antibody has been modified suchthat the antigen binding site binds to more than one antigen, such as adual function antibody which binds both HER3 and another antigen (e.g.,HER1, HER2, and HER4). In another embodiment, the invention pertains toa dual function antibody that targets antigens having the sameconformation, for example an antigen that has the same conformation ofHER3 in the “closed” or “inactive” state. Examples of antigens with thesame conformation of HER3 in the “closed” or “inactive” state include,but are not limited to, HER1 and HER4. Thus, a dual function antibodymay bind to both HER3 and HER1; HER3 and HER4, or HER1 and HER4. Thedual binding specificity of the dual function antibody may furthertranslate into dual activity, or inhibition of activity. (See e.g.,Jenny Bostrom et al., (2009) Science: 323; 1610-1614).

Antibodies with Extended Half Life

The present invention provides for antibodies that specifically bind toHER3 protein which have an extended half-life in vivo.

Many factors may affect a protein's half life in vivo. For examples,kidney filtration, metabolism in the liver, degradation by proteolyticenzymes (proteases), and immunogenic responses (e.g., proteinneutralization by antibodies and uptake by macrophages and dentriticcells). A variety of strategies can be used to extend the half life ofthe antibodies of the present invention. For example, by chemicallinkage to polyethyleneglycol (PEG), reCODE PEG, antibody scaffold,polysialic acid (PSA), hydroxyethyl starch (HES), albumin-bindingligands, and carbohydrate shields; by genetic fusion to proteins bindingto serum proteins, such as albumin, IgG, FcRn, and transferring; bycoupling (genetically or chemically) to other binding moieties that bindto serum proteins, such as nanobodies, Fabs, DARPins, avimers,affibodies, and anticalins; by genetic fusion to rPEG, albumin, domainof albumin, albumin-binding proteins, and Fc; or by incorporation intonanocarriers, slow release formulations, or medical devices.

To prolong the serum circulation of antibodies in vivo, inert polymermolecules such as high molecular weight PEG can be attached to theantibodies or a fragment thereof with or without a multifunctionallinker either through site-specific conjugation of the PEG to the N- orC-terminus of the antibodies or via epsilon-amino groups present onlysine residues. To pegylate an antibody, the antibody, or fragmentthereof, typically is reacted with polyethylene glycol (PEG), such as areactive ester or aldehyde derivative of PEG, under conditions in whichone or more PEG groups become attached to the antibody or antibodyfragment. The pegylation can be carried out by an acylation reaction oran alkylation reaction with a reactive PEG molecule (or an analogousreactive water-soluble polymer). As used herein, the term “polyethyleneglycol” is intended to encompass any of the forms of PEG that have beenused to derivatize other proteins, such as mono (C1-C10) alkoxy- oraryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certainembodiments, the antibody to be pegylated is an aglycosylated antibody.Linear or branched polymer derivatization that results in minimal lossof biological activity will be used. The degree of conjugation can beclosely monitored by SDS-PAGE and mass spectrometry to ensure properconjugation of PEG molecules to the antibodies. Unreacted PEG can beseparated from antibody-PEG conjugates by size-exclusion or byion-exchange chromatography. PEG-derivatized antibodies can be testedfor binding activity as well as for in vivo efficacy using methodswell-known to those of skill in the art, for example, by immunoassaysdescribed herein. Methods for pegylating proteins are known in the artand can be applied to the antibodies of the invention. See for example,EP 0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikawa et al.

Other modified pegylation technologies include reconstituting chemicallyorthogonal directed engineering technology (ReCODE PEG), whichincorporates chemically specified side chains into biosynthetic proteinsvia a reconstituted system that includes tRNA synthetase and tRNA. Thistechnology enables incorporation of more than 30 new amino acids intobiosynthetic proteins in E. coli, yeast, and mammalian cells. The tRNAincorporates a nonnative amino acid any place an amber codon ispositioned, converting the amber from a stop codon to one that signalsincorporation of the chemically specified amino acid.

Recombinant pegylation technology (rPEG) can also be used for serumhalf-life extension. This technology involves genetically fusing a300-600 amino acid unstructured protein tail to an existingpharmaceutical protein. Because the apparent molecular weight of such anunstructured protein chain is about 15-fold larger than its actualmolecular weight, the serum half-life of the protein is greatlyincreased. In contrast to traditional PEGylation, which requireschemical conjugation and repurification, the manufacturing process isgreatly simplified and the product is homogeneous.

Polysialytion is another technology, which uses the natural polymerpolysialic acid (PSA) to prolong the active life and improve thestability of therapeutic peptides and proteins. PSA is a polymer ofsialic acid (a sugar). When used for protein and therapeutic peptidedrug delivery, polysialic acid provides a protective microenvironment onconjugation. This increases the active life of the therapeutic proteinin the circulation and prevents it from being recognized by the immunesystem. The PSA polymer is naturally found in the human body. It wasadopted by certain bacteria which evolved over millions of years to coattheir walls with it. These naturally polysialylated bacteria were thenable, by virtue of molecular mimicry, to foil the body's defense system.PSA, nature's ultimate stealth technology, can be easily produced fromsuch bacteria in large quantities and with predetermined physicalcharacteristics. Bacterial PSA is completely non-immunogenic, even whencoupled to proteins, as it is chemically identical to PSA in the humanbody.

Another technology include the use of hydroxyethyl starch (“HES”)derivatives linked to antibodies. HES is a modified natural polymerderived from waxy maize starch and can be metabolized by the body'senzymes. HES solutions are usually administered to substitute deficientblood volume and to improve the rheological properties of the blood.Hesylation of an antibody enables the prolongation of the circulationhalf-life by increasing the stability of the molecule, as well as byreducing renal clearance, resulting in an increased biological activity.By varying different parameters, such as the molecular weight of HES, awide range of HES antibody conjugates can be customized.

Antibodies having an increased half-life in vivo can also be generatedintroducing one or more amino acid modifications (i.e., substitutions,insertions or deletions) into an IgG constant domain, or FcRn bindingfragment thereof (preferably a Fc or hinge Fc domain fragment). See,e.g., International Publication No. WO 98/23289; InternationalPublication No. WO 97/34631; and U.S. Pat. No. 6,277,375.

Further, antibodies can be conjugated to albumin in order to make theantibody or antibody fragment more stable in vivo or have a longer halflife in vivo. The techniques are well-known in the art, see, e.g.,International Publication Nos. WO 93/15199, WO 93/15200, and WO01/77137; and European Patent No. EP 413,622.

The HER3 antibody or a fragment thereof may also be fused to one or morehuman serum albumin (HSA) polypeptides, or a portion thereof. HSA, aprotein of 585 amino acids in its mature form, is responsible for asignificant proportion of the osmotic pressure of serum and alsofunctions as a carrier of endogenous and exogenous ligands. The role ofalbumin as a carrier molecule and its inert nature are desirableproperties for use as a carrier and transporter of polypeptides in vivo.The use of albumin as a component of an albumin fusion protein as acarrier for various proteins has been suggested in WO 93/15199, WO93/15200, and EP 413 622. The use of N-terminal fragments of HSA forfusions to polypeptides has also been proposed (EP 399 666).Accordingly, by genetically or chemically fusing or conjugating theantibodies or fragments thereof to albumin, can stabilize or extend theshelf-life, and/or to retain the molecule's activity for extendedperiods of time in solution, in vitro and/or in vivo.

Fusion of albumin to another protein may be achieved by geneticmanipulation, such that the DNA coding for HSA, or a fragment thereof,is joined to the DNA coding for the protein. A suitable host is thentransformed or transfected with the fused nucleotide sequences, soarranged on a suitable plasmid as to express a fusion polypeptide. Theexpression may be effected in vitro from, for example, prokaryotic oreukaryotic cells, or in vivo e.g. from a transgenic organism. Additionalmethods pertaining to HSA fusions can be found, for example, in WO2001077137 and WO 200306007, incorporated herein by reference. In aspecific embodiment, the expression of the fusion protein is performedin mammalian cell lines, for example, CHO cell lines. Altereddifferential binding of an antibody to a receptor at low or high pHs isalso contemplated to be within the scope of the invention. For example,the affinity of an antibody may be modified such that it remains boundto it's receptor at a low pH, e.g., the low pH within a lyzozome, bymodifying the antibody to include additional amino acids such as ahistine in a CDR of the antibody (See e.g., Tomoyuki Igawa et al. (2010)Nature Biotechnology; 28, 1203-1207).

Antibody Conjugates

The present invention provides antibodies or fragments thereof thatspecifically bind to a HER3 protein recombinantly fused or chemicallyconjugated (including both covalent and non-covalent conjugations) to aheterologous protein or polypeptide (or fragment thereof, preferably toa polypeptide of at least 10, at least 20, at least 30, at least 40, atleast 50, at least 60, at least 70, at least 80, at least 90 or at least100 amino acids) to generate fusion proteins. In particular, theinvention provides fusion proteins comprising an antibody fragmentdescribed herein (e.g., a Fab fragment, Fd fragment, Fv fragment, F(ab)2fragment, a VH domain, a VH CDR, a VL domain or a VL CDR) and aheterologous protein, polypeptide, or peptide. Methods for fusing orconjugating proteins, polypeptides, or peptides to an antibody or anantibody fragment are known in the art. See, e.g., U.S. Pat. Nos.5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5,112,946;European Patent Nos. EP 307,434 and EP 367,166; InternationalPublication Nos. WO 96/04388 and WO 91/06570; Ashkenazi et al., (1991)Proc. Natl. Acad. Sci. USA 88:10535-10539; Zheng et al., (1995) J.Immunol. 154:5590-5600; and Vil et al., (1992) Proc. Natl. Acad. Sci.USA 89:11337-11341.

Additional fusion proteins may be generated through the techniques ofgene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling(collectively referred to as “DNA shuffling”). DNA shuffling may beemployed to alter the activities of antibodies of the invention orfragments thereof (e.g., antibodies or fragments thereof with higheraffinities and lower dissociation rates). See, generally, U.S. Pat. Nos.5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten etal., (1997) Curr. Opinion Biotechnol. 8:724-33; Harayama, (1998) TrendsBiotechnol. 16(2):76-82; Hansson et al., (1999) J. Mol. Biol.287:265-76; and Lorenzo and Blasco, (1998) Biotechniques 24(2):308-313(each of these patents and publications are hereby incorporated byreference in its entirety). Antibodies or fragments thereof, or theencoded antibodies or fragments thereof, may be altered by beingsubjected to random mutagenesis by error-prone PCR, random nucleotideinsertion or other methods prior to recombination. A polynucleotideencoding an antibody or fragment thereof that specifically binds to aHER3 protein may be recombined with one or more components, motifs,sections, parts, domains, fragments, etc. of one or more heterologousmolecules. Moreover, the antibodies or fragments thereof can be fused tomarker sequences, such as a peptide to facilitate purification. Inpreferred embodiments, the marker amino acid sequence is ahexa-histidine (SEQ ID NO: 495) peptide, such as the tag provided in apQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311),among others, many of which are commercially available. As described inGentz et al., (1989) Proc. Natl. Acad. Sci. USA 86:821-824, forinstance, hexa-histidine (SEQ ID NO: 495) provides for convenientpurification of the fusion protein. Other peptide tags useful forpurification include, but are not limited to, the hemagglutinin (“HA”)tag, which corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson et al., (1984) Cell 37:767), and the“flag” tag.

In other embodiments, antibodies of the present invention or fragmentsthereof conjugated to a diagnostic or detectable agent. Such antibodiescan be useful for monitoring or prognosing the onset, development,progression and/or severity of a disease or disorder as part of aclinical testing procedure, such as determining the efficacy of aparticular therapy. Such diagnosis and detection can accomplished bycoupling the antibody to detectable substances including, but notlimited to, various enzymes, such as, but not limited to, horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; prosthetic groups, such as, but not limited to,streptavidinlbiotin and avidin/biotin; fluorescent materials, such as,but not limited to, umbelliferone, fluorescein, fluoresceinisothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; luminescent materials, such as, but notlimited to, luminol; bioluminescent materials, such as but not limitedto, luciferase, luciferin, and aequorin; radioactive materials, such as,but not limited to, iodine (¹³¹I, ¹²⁵I, ¹²³I, and ¹²¹I), carbon (¹⁴C),sulfur (³⁵S), tritium (³H), indium (¹¹⁵In, ¹¹³In, ¹¹²In, and ¹¹¹In),technetium (⁹⁹Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium(¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu,¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr,¹⁰⁵Rh, ⁹⁷Ru, ⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se,¹¹³Sn, and ¹¹⁷Tin; and positron emitting metals using various positronemission tomographies, and noradioactive paramagnetic metal ions.

The present invention further encompasses uses of antibodies orfragments thereof conjugated to a therapeutic moiety. An antibody orfragment thereof may be conjugated to a therapeutic moiety such as acytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent ora radioactive metal ion, e.g., alpha-emitters. A cytotoxin or cytotoxicagent includes any agent that is detrimental to cells.

Further, an antibody or fragment thereof may be conjugated to atherapeutic moiety or drug moiety that modifies a given biologicalresponse. Therapeutic moieties or drug moieties are not to be construedas limited to classical chemical therapeutic agents. For example, thedrug moiety may be a protein, peptide, or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, cholera toxin, ordiphtheria toxin; a protein such as tumor necrosis factor, α-interferon,β-interferon, nerve growth factor, platelet derived growth factor,tissue plasminogen activator, an apoptotic agent, an anti-angiogenicagent; or, a biological response modifier such as, for example, alymphokine. In one embodiment, the anti-HER3 antibody, or a fragmentthereof, conjugated to a therapeutic moiety, such as a cytotoxin, a drug(e.g., an immunosuppressant) or a radiotoxin. Such conjugates arereferred to herein as “immunoconjugates”. Immunoconjugates that includeone or more cytotoxins are referred to as “immunotoxins.” A cytotoxin orcytotoxic agent includes any agent that is detrimental to (e.g., kills)cells. Examples include taxon, cytochalasin B, gramicidin D, ethidiumbromide, emetine, mitomycin, etoposide, tenoposide, vincristine,vinblastine, t. colchicin, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof. Therapeuticagents also include, for example, antimetabolites (e.g., methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine), ablating agents (e.g., mechlorethamine, thioepachloraxnbucil, meiphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin, anthracyclines(e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics(e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, andanthramycin (AMC)), and anti-mitotic agents (e.g., vincristine andvinblastine). (See e.g., Seattle Genetics US20090304721).

Other examples of therapeutic cytotoxins that can be conjugated to anantibody of the invention include duocarmycins, calicheamicins,maytansines and auristatins, and derivatives thereof. An example of acalicheamicin antibody conjugate is commercially available (Mylotarg™;Wyeth-Ayerst).

Cytoxins can be conjugated to antibodies of the invention using linkertechnology available in the art. Examples of linker types that have beenused to conjugate a cytotoxin to an antibody include, but are notlimited to, hydrazones, thioethers, esters, disulfides andpeptide-containing linkers. A linker can be chosen that is, for example,susceptible to cleavage by low pH within the lysosomal compartment orsusceptible to cleavage by proteases, such as proteases preferentiallyexpressed in tumor tissue such as cathepsins (e.g., cathepsins B, C, D).

For further discussion of types of cytotoxins, linkers and methods forconjugating therapeutic agents to antibodies, see also Saito et al.,(2003) Adv. Drug Deliv. Rev. 55:199-215; Trail et al., (2003) CancerImmunol. Immunother. 52:328-337; Payne, (2003) Cancer Cell 3:207-212;Allen, (2002) Nat. Rev. Cancer 2:750-763; Pastan and Kreitman, (2002)Curr. Opin. Investig. Drugs 3:1089-1091; Senter and Springer, (2001)Adv. Drug Deliv. Rev. 53:247-264.

Antibodies of the present invention also can be conjugated to aradioactive isotope to generate cytotoxic radiopharmaceuticals, alsoreferred to as radioimmunoconjugates. Examples of radioactive isotopesthat can be conjugated to antibodies for use diagnostically ortherapeutically include, but are not limited to, iodine¹³¹, indium¹¹¹,yttrium⁹⁰, and lutetium¹⁷⁷, Method for preparing radioimmunconjugatesare established in the art. Examples of radioimmunoconjugates arecommercially available, including Zevalin™ (DEC Pharmaceuticals) andBexxar™ (Corixa Pharmaceuticals), and similar methods can be used toprepare radioimmunoconjugates using the antibodies of the invention. Incertain embodiments, the macrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA) whichcan be attached to the antibody via a linker molecule. Such linkermolecules are commonly known in the art and described in Denardo et al.,(1998) Clin Cancer Res. 4(10):2483-90; Peterson et al., (1999)Bioconjug. Chem. 10(4):553-7; and Zimmerman et al., (1999) Nucl. Med.Biol. 26(8):943-50, each incorporated by reference in their entireties.

Techniques for conjugating therapeutic moieties to antibodies are wellknown, see, e.g., Amon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies 84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., (1982)Immunol. Rev. 62:119-58.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

Antibody Combinations

An another aspect, the invention pertains to HER3 antibodies, orfragments thereof of the invention used with other therapeutic agentssuch as another antibodies, small molecule inhibitors, mTOR inhibitorsor PI3Kinase inhibitors. Examples include, but are not limited to, thefollowing:

HER1 inhibitors: The HER3 antibodies or fragments thereof can be usedwith HER1 inhibitors which include, but are not limited to, Matuzumab(EMD72000), Erbitux®/Cetuximab (Imclone), Vectibix®/Panitumumab (Amgen),mAb 806, and Nimotuzumab (TheraCIM), Iressa®/Gefitinib (Astrazeneca);CI-1033 (PD183805) (Pfizer), Lapatinib (GW-572016) (GlaxoSmithKline),Tykerb®/Lapatinib Ditosylate (SmithKlineBeecham), Tarceva®/Erlotinib HCL(OSI-774) (OSI Pharma), and PKI-166 (Novartis), andN-[4-[(3-Chloro-4-fluorophenyl)amino]-7-[[(3″S″)-tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4(dimethylamino)-2-butenamide,sold under the tradename Tovok® by Boehringer Ingelheim).

HER2 inhibitors: The HER3 antibodies or fragments thereof can be usedwith HER2 inhibitors which include, but are not limited to, Pertuzumab(sold under the trademark Omnitarg®, by Genentech), Trastuzumab (soldunder the trademark Herceptin® by Genentech/Roche), MM-111, neratinib(also known as HKI-272,(2E)-N-[4-[[3-chloro-4-[(pyridin-2-yl)methoxy]phenyl]amino]-3-cyano-7-ethoxyquinolin-6-yl]-4-(dimethylamino)but-2-enamide, and describedPCT Publication No. WO 05/028443), lapatinib or lapatinib ditosylate(sold under the trademark Tykerb® by GlaxoSmithKline.

HER3 inhibitors: The HER3 antibodies or fragments thereof can be usedwith HER3 inhibitors which include, but are not limited to, MM-121,MM-111, IB4C3, 2DID12 (U3 Pharma AG), AMG888 (Amgen), AV-203 (Aveo),MEHD7945A (Genentech), and small molecules that inhibit HER3.

HER4 inhibitors: The HER3 antibodies or fragments thereof can be usedwith HER4 inhibitors.

PI3K inhibitors: The HER3 antibodies or fragments thereof can be usedwith PI3 kinase inhibitors which include, but are not limited to,4-[2-(1H-Indazol-4-yl)-6-[[4-(methylsulfonyl)piperazin-1-yl]methyl]thieno[3,2-d]pyrimidin-4-yl]morpholine(also known as GDC 0941 and described in PCT Publication Nos. WO09/036082 and WO 09/055730),2-Methyl-2-[4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydroimidazo[4,5-c]quinolin-1-yl]phenyl]propionitrile(also known as BEZ 235 or NVP-BEZ 235, and described in PCT PublicationNo. WO 06/122806), BMK120 and BYL719.

mTOR inhibitors: The HER3 antibodies or fragments thereof can be usedwith mTOR inhibitors which include, but are not limited to, Temsirolimus(sold under the tradename Torisel® by Pfizer), ridaforolimus (formallyknown as deferolimus, (1R,2R,4S)-4-[(2R)-2[(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.04,9]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyldimethylphosphinate, also known as Deforolimus, AP23573 and MK8669(Ariad Pharm.), and described in PCT Publication No. WO 03/064383),everolimus (RAD001) (sold under the tradename Afinitor® by Novartis),One or more therapeutic agents may be administered either simultaneouslyor before or after administration of a HER3 antibody or fragment thereofof the present invention.

Methods of Producing Antibodies of the Invention

(i) Nucleic Acids Encoding the Antibodies

The invention provides substantially purified nucleic acid moleculeswhich encode polypeptides comprising segments or domains of theHER3-binding antibody chains described above. Some of the nucleic acidsof the invention comprise the nucleotide sequence encoding the HER3antibody heavy chain variable region, and/or the nucleotide sequenceencoding the light chain variable region. In a specific embodiment, thenucleic acid molecules are those identified in Table 1. Some othernucleic acid molecules of the invention comprise nucleotide sequencesthat are substantially identical (e.g., at least 65, 80%, 95%, or 99%)to the nucleotide sequences of those identified in Table 1. Whenexpressed from appropriate expression vectors, polypeptides encoded bythese polynucleotides are capable of exhibiting HER3 antigen bindingcapacity.

Also provided in the invention are polynucleotides which encode at leastone CDR region and usually all three CDR regions from the heavy or lightchain of the HER3-binding antibody set forth above. Some otherpolynucleotides encode all or substantially all of the variable regionsequence of the heavy chain and/or the light chain of the HER3-bindingantibody set forth above. Because of the degeneracy of the code, avariety of nucleic acid sequences will encode each of the immunoglobulinamino acid sequences.

The nucleic acid molecules of the invention can encode both a variableregion and a constant region of the antibody. Some of nucleic acidsequences of the invention comprise nucleotides encoding a mature heavychain variable region sequence that is substantially identical (e.g., atleast 80%, 90%, or 99%) to the mature heavy chain variable regionsequence of a HER3 antibody set forth in Table 1. Some other nucleicacid sequences comprising nucleotide encoding a mature light chainvariable region sequence that is substantially identical (e.g., at least80%, 90%, or 99%) to the mature light chain variable region sequence ofa HER3 antibody set forth in Table 1.

The polynucleotide sequences can be produced by de novo solid-phase DNAsynthesis or by PCR mutagenesis of an existing sequence (e.g., sequencesas described in the Examples below) encoding an HER3-binding antibody orits binding fragment. Direct chemical synthesis of nucleic acids can beaccomplished by methods known in the art, such as the phosphotriestermethod of Narang et al., (1979) Meth. Enzymol. 68:90; the phosphodiestermethod of Brown et al., (1979) Meth. Enzymol. 68:109; thediethylphosphoramidite method of Beaucage et al., (1981) Tetra. Lett.,22:1859; and the solid support method of U.S. Pat. No. 4,458,066.Introducing mutations to a polynucleotide sequence by PCR can beperformed as described in, e.g., PCR Technology: Principles andApplications for DNA Amplification, H. A. Erlich (Ed.), Freeman Press,NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Inniset al. (Ed.), Academic Press, San Diego, Calif., 1990; Mattila et al.,(1991) Nucleic Acids Res. 19:967; and Eckert et al., (1991) PCR Methodsand Applications 1:17.

Also provided in the invention are expression vectors and host cells forproducing the HER3-binding antibodies described above. Variousexpression vectors can be employed to express the polynucleotidesencoding the HER3-binding antibody chains or binding fragments. Bothviral-based and nonviral expression vectors can be used to produce theantibodies in a mammalian host cell. Nonviral vectors and systemsinclude plasmids, episomal vectors, typically with an expressioncassette for expressing a protein or RNA, and human artificialchromosomes (see, e.g., Harrington et al., (1997) Nat Genet 15:345). Forexample, nonviral vectors useful for expression of the HER3-bindingpolynucleotides and polypeptides in mammalian (e.g., human) cellsinclude pThioHis A, B & C, pcDNA3.1/His, pEBVHis A, B & C, (Invitrogen,San Diego, Calif.), MPSV vectors, and numerous other vectors known inthe art for expressing other proteins. Useful viral vectors includevectors based on retroviruses, adenoviruses, adenoassociated viruses,herpes viruses, vectors based on SV40, papilloma virus, HBP Epstein Barrvirus, vaccinia virus vectors and Semliki Forest virus (SFV). See, Brentet al., (1995) supra; Smith, Annu. Rev. Microbiol. 49:807; and Rosenfeldet al., (1992) Cell 68:143.

The choice of expression vector depends on the intended host cells inwhich the vector is to be expressed. Typically, the expression vectorscontain a promoter and other regulatory sequences (e.g., enhancers) thatare operably linked to the polynucleotides encoding an HER3-bindingantibody chain or fragment. In some embodiments, an inducible promoteris employed to prevent expression of inserted sequences except underinducing conditions. Inducible promoters include, e.g., arabinose, lacZ,metallothionein promoter or a heat shock promoter. Cultures oftransformed organisms can be expanded under noninducing conditionswithout biasing the population for coding sequences whose expressionproducts are better tolerated by the host cells. In addition topromoters, other regulatory elements may also be required or desired forefficient expression of an HER3-binding antibody chain or fragment.These elements typically include an ATG initiation codon and adjacentribosome binding site or other sequences. In addition, the efficiency ofexpression may be enhanced by the inclusion of enhancers appropriate tothe cell system in use (see, e.g., Scharf et al., (1994) Results Probl.Cell Differ. 20:125; and Bittner et al., (1987) Meth. Enzymol.,153:516). For example, the SV40 enhancer or CMV enhancer may be used toincrease expression in mammalian host cells.

The expression vectors may also provide a secretion signal sequenceposition to form a fusion protein with polypeptides encoded by insertedHER3-binding antibody sequences. More often, the inserted HER3-bindingantibody sequences are linked to a signal sequences before inclusion inthe vector. Vectors to be used to receive sequences encodingHER3-binding antibody light and heavy chain variable domains sometimesalso encode constant regions or parts thereof. Such vectors allowexpression of the variable regions as fusion proteins with the constantregions thereby leading to production of intact antibodies or fragmentsthereof. Typically, such constant regions are human.

The host cells for harboring and expressing the HER3-binding antibodychains can be either prokaryotic or eukaryotic. E. coli is oneprokaryotic host useful for cloning and expressing the polynucleotidesof the present invention. Other microbial hosts suitable for use includebacilli, such as Bacillus subtilis, and other enterobacteriaceae, suchas Salmonella, Serratia, and various Pseudomonas species. In theseprokaryotic hosts, one can also make expression vectors, which typicallycontain expression control sequences compatible with the host cell(e.g., an origin of replication). In addition, any number of a varietyof well-known promoters will be present, such as the lactose promotersystem, a tryptophan (trp) promoter system, a beta-lactamase promotersystem, or a promoter system from phage lambda. The promoters typicallycontrol expression, optionally with an operator sequence, and haveribosome binding site sequences and the like, for initiating andcompleting transcription and translation. Other microbes, such as yeast,can also be employed to express HER3-binding polypeptides of theinvention. Insect cells in combination with baculovirus vectors can alsobe used. In some preferred embodiments, mammalian host cells are used toexpress and produce the HER3-binding polypeptides of the presentinvention. For example, they can be either a hybridoma cell lineexpressing endogenous immunoglobulin genes (e.g., the 1D6.C9 myelomahybridoma clone as described in the Examples) or a mammalian cell lineharboring an exogenous expression vector (e.g., the SP2/0 myeloma cellsexemplified below). These include any normal mortal or normal orabnormal immortal animal or human cell. For example, a number ofsuitable host cell lines capable of secreting intact immunoglobulinshave been developed including the CHO cell lines, various Cos celllines, HeLa cells, myeloma cell lines, transformed B-cells andhybridomas. The use of mammalian tissue cell culture to expresspolypeptides is discussed generally in, e.g., Winnacker, FROM GENES TOCLONES, VCH Publishers, N.Y., N.Y., 1987. Expression vectors formammalian host cells can include expression control sequences, such asan origin of replication, a promoter, and an enhancer (see, e.g., Queenet al., (1986) Immunol. Rev. 89:49-68), and necessary processinginformation sites, such as ribosome binding sites, RNA splice sites,polyadenylation sites, and transcriptional terminator sequences. Theseexpression vectors usually contain promoters derived from mammaliangenes or from mammalian viruses. Suitable promoters may be constitutive,cell type-specific, stage-specific, and/or modulatable or regulatable.Useful promoters include, but are not limited to, the metallothioneinpromoter, the constitutive adenovirus major late promoter, thedexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP polIIIpromoter, the constitutive MPSV promoter, the tetracycline-inducible CMVpromoter (such as the human immediate-early CMV promoter), theconstitutive CMV promoter, and promoter-enhancer combinations known inthe art.

Methods for introducing expression vectors containing the polynucleotidesequences of interest vary depending on the type of cellular host. Forexample, calcium chloride transfection is commonly utilized forprokaryotic cells, whereas calcium phosphate treatment orelectroporation may be used for other cellular hosts. (See generallySambrook, et al., supra). Other methods include, e.g., electroporation,calcium phosphate treatment, liposome-mediated transformation, injectionand microinjection, ballistic methods, virosomes, immunoliposomes,polycation:nucleic acid conjugates, naked DNA, artificial virions,fusion to the herpes virus structural protein VP22 (Elliot and O'Hare,(1997) Cell 88:223), agent-enhanced uptake of DNA, and ex vivotransduction. For long-term, high-yield production of recombinantproteins, stable expression will often be desired. For example, celllines which stably express HER3-binding antibody chains or bindingfragments can be prepared using expression vectors of the inventionwhich contain viral origins of replication or endogenous expressionelements and a selectable marker gene. Following the introduction of thevector, cells may be allowed to grow for 1-2 days in an enriched mediabefore they are switched to selective media. The purpose of theselectable marker is to confer resistance to selection, and its presenceallows growth of cells which successfully express the introducedsequences in selective media. Resistant, stably transfected cells can beproliferated using tissue culture techniques appropriate to the celltype.

(ii) Generation of Monoclonal Antibodies of the Invention

Monoclonal antibodies (mAbs) can be produced by a variety of techniques,including conventional monoclonal antibody methodology e.g., thestandard somatic cell hybridization technique of Kohler and Milstein,(1975) Nature 256:495. Many techniques for producing monoclonal antibodycan be employed e.g., viral or oncogenic transformation of Blymphocytes.

An animal system for preparing hybridomas is the murine system.Hybridoma production in the mouse is a well established procedure.Immunization protocols and techniques for isolation of immunizedsplenocytes for fusion are known in the art. Fusion partners (e.g.,murine myeloma cells) and fusion procedures are also known.

Chimeric or humanized antibodies of the present invention can beprepared based on the sequence of a murine monoclonal antibody preparedas described above. DNA encoding the heavy and light chainimmunoglobulins can be obtained from the murine hybridoma of interestand engineered to contain non-murine (e.g., human) immunoglobulinsequences using standard molecular biology techniques. For example, tocreate a chimeric antibody, the murine variable regions can be linked tohuman constant regions using methods known in the art (see e.g., U.S.Pat. No. 4,816,567 to Cabilly et al.). To create a humanized antibody,the murine CDR regions can be inserted into a human framework usingmethods known in the art. See e.g., U.S. Pat. No. 5,225,539 to Winter,and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 toQueen et al.

In a certain embodiment, the antibodies of the invention are humanmonoclonal antibodies. Such human monoclonal antibodies directed againstHER3 can be generated using transgenic or transchromosomic mice carryingparts of the human immune system rather than the mouse system. Thesetransgenic and transchromosomic mice include mice referred to herein asHuMAb mice and KM mice, respectively, and are collectively referred toherein as “human Ig mice.”

The HuMAb Mouse® (Medarex, Inc.) contains human immunoglobulin geneminiloci that encode un-rearranged human heavy (μ and γ) and κ lightchain immunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (see e.g., Lonberg et al.,(1994) Nature 368(6474): 856-859). Accordingly, the mice exhibit reducedexpression of mouse IgM or κ, and in response to immunization, theintroduced human heavy and light chain transgenes undergo classswitching and somatic mutation to generate high affinity human IgGκmonoclonal (Lonberg et al., (1994) supra; reviewed in Lonberg, (1994)Handbook of Experimental Pharmacology 113:49-101; Lonberg and Huszar,(1995) Intern. Rev. Immunol. 13:65-93, and Harding and Lonberg, (1995)Ann. N. Y. Acad. Sci. 764:536-546). The preparation and use of HuMAbmice, and the genomic modifications carried by such mice, is furtherdescribed in Taylor et al., (1992) Nucleic Acids Research 20:6287-6295;Chen et al., (1993) International Immunology 5:647-656; Tuaillon et al.,(1993) Proc. Natl. Acad. Sci. USA 94:3720-3724; Choi et al., (1993)Nature Genetics 4:117-123; Chen et al., (1993) EMBO J. 12:821-830;Tuaillon et al., (1994) J. Immunol. 152:2912-2920; Taylor et al., (1994)International Immunology 579-591; and Fishwild et al., (1996) NatureBiotechnology 14:845-851, the contents of all of which are herebyspecifically incorporated by reference in their entirety. See further,U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650;5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429; all toLonberg and Kay; U.S. Pat. No. 5,545,807 to Surani et al.; PCTPublication Nos. WO 92103918, WO 93/12227, WO 94/25585, WO 97113852, WO98/24884 and WO 99/45962, all to Lonberg and Kay; and PCT PublicationNo. WO 01/14424 to Korman et al.

In another embodiment, human antibodies of the invention can be raisedusing a mouse that carries human immunoglobulin sequences on transgenesand transchomosomes such as a mouse that carries a human heavy chaintransgene and a human light chain trans chromosome. Such mice, referredto herein as “KM mice”, are described in detail in PCT Publication WO02/43478 to Ishida et al.

Still further, alternative transgenic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseHER3-binding antibodies of the invention. For example, an alternativetransgenic system referred to as the Xenomouse (Abgenix, Inc.) can beused. Such mice are described in, e.g., U.S. Pat. Nos. 5,939,598;6,075,181; 6,114,598; 6, 150,584 and 6,162,963 to Kucherlapati et al.

Moreover, alternative transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseHER3-binding antibodies of the invention. For example, mice carryingboth a human heavy chain transchromosome and a human light chaintranchromosome, referred to as “TC mice” can be used; such mice aredescribed in Tomizuka et al., (2000) Proc. Natl. Acad. Sci. USA97:722-727. Furthermore, cows carrying human heavy and light chaintranschromosomes have been described in the art (Kuroiwa et al., (2002)Nature Biotechnology 20:889-894) and can be used to raise HER3-bindingantibodies of the invention.

Human monoclonal antibodies of the invention can also be prepared usingphage display methods for screening libraries of human immunoglobulingenes. Such phage display methods for isolating human antibodies areestablished in the art or described in the examples below. See forexample: U.S. Pat. Nos. 5,223,409; 5,403,484; and U.S. Pat. No.5,571,698 to Ladner et al.; U.S. Pat. Nos. 5,427,908 and 5,580,717 toDower et al.; U.S. Pat. Nos. 5,969,108 and 6,172,197 to McCafferty etal.; and U.S. Pat. Nos. 5,885,793; 6,521,404; 6,544,731; 6,555,313;6,582,915 and 6,593,081 to Griffiths et al.

Human monoclonal antibodies of the invention can also be prepared usingSCID mice into which human immune cells have been reconstituted suchthat a human antibody response can be generated upon immunization. Suchmice are described in, for example, U.S. Pat. Nos. 5,476,996 and5,698,767 to Wilson et al.

(iii) Framework or Fc Engineering

Engineered antibodies of the invention include those in whichmodifications have been made to framework residues within VH and/or VL,e.g. to improve the properties of the antibody. Typically such frameworkmodifications are made to decrease the immunogenicity of the antibody.For example, one approach is to “backmutate” one or more frameworkresidues to the corresponding germline sequence. More specifically, anantibody that has undergone somatic mutation may contain frameworkresidues that differ from the germline sequence from which the antibodyis derived. Such residues can be identified by comparing the antibodyframework sequences to the germline sequences from which the antibody isderived. To return the framework region sequences to their germlineconfiguration, the somatic mutations can be “backmutated” to thegermline sequence by, for example, site-directed mutagenesis. Such“backmutated” antibodies are also intended to be encompassed by theinvention.

Another type of framework modification involves mutating one or moreresidues within the framework region, or even within one or more CDRregions, to remove T cell-epitopes to thereby reduce the potentialimmunogenicity of the antibody. This approach is also referred to as“deimmunization” and is described in further detail in U.S. PatentPublication No. 20030153043 by Carr et al.

In addition or alternative to modifications made within the framework orCDR regions, antibodies of the invention may be engineered to includemodifications within the Fc region, typically to alter one or morefunctional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding, and/or antigen-dependentcellular cytotoxicity. Furthermore, an antibody of the invention may bechemically modified (e.g., one or more chemical moieties can be attachedto the antibody) or be modified to alter its glycosylation, again toalter one or more functional properties of the antibody. Each of theseembodiments is described in further detail below. The numbering ofresidues in the Fc region is that of the EU index of Kabat.

In one embodiment, the hinge region of CH1 is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further in U.S. Pat.No. 5,677,425 by Bodmer et al. The number of cysteine residues in thehinge region of CH1 is altered to, for example, facilitate assembly ofthe light and heavy chains or to increase or decrease the stability ofthe antibody.

In another embodiment, the Fc hinge region of an antibody is mutated todecrease the biological half-life of the antibody. More specifically,one or more amino acid mutations are introduced into the CH2-CH3 domaininterface region of the Fc-hinge fragment such that the antibody hasimpaired Staphylococcyl protein A (SpA) binding relative to nativeFc-hinge domain SpA binding. This approach is described in furtherdetail in U.S. Pat. No. 6,165,745 by Ward et al.

In yet other embodiments, the Fc region is altered by replacing at leastone amino acid residue with a different amino acid residue to alter theeffector functions of the antibody. For example, one or more amino acidscan be replaced with a different amino acid residue such that theantibody has an altered affinity for an effector ligand but retains theantigen-binding ability of the parent antibody. The effector ligand towhich affinity is altered can be, for example, an Fc receptor or the C1component of complement. This approach is described in further detail inU.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.

In another embodiment, one or more amino acids selected from amino acidresidues can be replaced with a different amino acid residue such thatthe antibody has altered C1q binding and/or reduced or abolishedcomplement dependent cytotoxicity (CDC). This approach is described infurther detail in U.S. Pat. No. 6,194,551 by Idusogie et al.

In another embodiment, one or more amino acid residues are altered tothereby alter the ability of the antibody to fix complement. Thisapproach is described further in PCT Publication WO 94/29351 by Bodmeret al.

In yet another embodiment, the Fc region is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to increase the affinity of the antibody foran Fcγ receptor by modifying one or more amino acids. This approach isdescribed further in PCT Publication WO 00/42072 by Presta. Moreover,the binding sites on human IgG1 for FcγR1, FcγRII, FcγRIII and FcRn havebeen mapped and variants with improved binding have been described (seeShields et al., (2001) J. Biol. Chen. 276:6591-6604).

In still another embodiment, the glycosylation of an antibody ismodified. For example, an aglycoslated antibody can be made (i.e., theantibody lacks glycosylation). Glycosylation can be altered to, forexample, increase the affinity of the antibody for “antigen’. Suchcarbohydrate modifications can be accomplished by, for example, alteringone or more sites of glycosylation within the antibody sequence. Forexample, one or more amino acid substitutions can be made that result inelimination of one or more variable region framework glycosylation sitesto thereby eliminate glycosylation at that site. Such aglycosylation mayincrease the affinity of the antibody for antigen. Such an approach isdescribed in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 byCo et al.

Additionally or alternatively, an antibody can be made that has analtered type of glycosylation, such as a hypofucosylated antibody havingreduced amounts of fucosyl residues or an antibody having increasedbisecting GlcNac structures. Such altered glycosylation patterns havebeen demonstrated to increase the ADCC ability of antibodies. Suchcarbohydrate modifications can be accomplished by, for example,expressing the antibody in a host cell with altered glycosylationmachinery. Cells with altered glycosylation machinery have beendescribed in the art and can be used as host cells in which to expressrecombinant antibodies of the invention to thereby produce an antibodywith altered glycosylation. For example, EP 1,176,195 by Hang et al.describes a cell line with a functionally disrupted FUT8 gene, whichencodes a fucosyl transferase, such that antibodies expressed in such acell line exhibit hypofucosylation. PCT Publication WO 03/035835 byPresta describes a variant CHO cell line, Lecl3 cells, with reducedability to attach fucose to Asn(297)-linked carbohydrates, alsoresulting in hypofucosylation of antibodies expressed in that host cell(see also Shields et al., (2002) J. Biol. Chem. 277:26733-26740). PCTPublication WO 99/54342 by Umana et al. describes cell lines engineeredto express glycoprotein-modifying glycosyl transferases (e.g.,beta(1,4)-N acetylglucosaminyltransferase III (GnTIII)) such thatantibodies expressed in the engineered cell lines exhibit increasedbisecting GlcNac structures which results in increased ADCC activity ofthe antibodies (see also Umana et al., (1999) Nat. Biotech. 17:176-180).

In another embodiment, the antibody is modified to increase itsbiological half-life. Various approaches are possible. For example, oneor more of the following mutations can be introduced: T252L, T254S,T256F, as described in U.S. Pat. No. 6,277,375 to Ward. Alternatively,to increase the biological half life, the antibody can be altered withinthe CH1 or CL region to contain a salvage receptor binding epitope takenfrom two loops of a CH2 domain of an Fc region of an IgG, as describedin U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al.

(iv) Methods of Engineering Altered Antibodies

As discussed above, the HER3-binding antibodies having VH and VLsequences or full length heavy and light chain sequences shown hereincan be used to create new HER3-binding antibodies by modifying fulllength heavy chain and/or light chain sequences, VH and/or VL sequences,or the constant region(s) attached thereto. Thus, in another aspect ofthe invention, the structural features of a HER3-binding antibody of theinvention are used to create structurally related HER3-bindingantibodies that retain at least one functional property of theantibodies of the invention, such as binding to human HER3 and alsoinhibiting one or more functional properties of HER3 For example, one ormore CDR regions of the antibodies of the present invention, ormutations thereof, can be combined recombinantly with known frameworkregions and/or other CDRs to create additional,recombinantly-engineered, HER3-binding antibodies of the invention, asdiscussed above. Other types of modifications include those described inthe previous section. The starting material for the engineering methodis one or more of the VH and/or VL sequences provided herein, or one ormore CDR regions thereof. To create the engineered antibody, it is notnecessary to actually prepare (i.e., express as a protein) an antibodyhaving one or more of the VH and/or VL sequences provided herein, or oneor more CDR regions thereof. Rather, the information contained in thesequence(s) is used as the starting material to create a “secondgeneration” sequence(s) derived from the original sequence(s) and thenthe “second generation” sequence(s) is prepared and expressed as aprotein.

Accordingly, in another embodiment, the invention provides a method forpreparing a HER3-binding antibody consisting of: a heavy chain variableregion antibody sequence having a CDR1 sequence selected from the groupconsisting of SEQ ID NOs: 2, 8, 20, 26, 38, 44, 56, 62, 74, 80, 92, 98,110, 116, 128, 134, 146, 152, 164, 170, 182, 188, 200, 206, 218, 224,236, 242, 254, 260, 272, 278, 290, 296, 308, 314, 326, 332, 344, 350,362, and 368; a CDR2 sequence selected from the group consisting of SEQID NOs: 3, 9, 21, 27, 39, 45, 57, 63, 75, 81, 93, 99, 111, 117, 129,135, 147, 153, 165, 171, 183, 189, 201, 207, 219, 225, 237, 243, 255,261, 273, 279, 291, 297, 309, 315, 327, 333, 345, 351, 363, and 369;and/or a CDR3 sequence selected from the group consisting of SEQ ID NOs:4, 10, 22, 28, 40, 46, 58, 64, 75, 82, 94, 100, 112, 118, 130, 136, 148,154, 166, 172, 184, 190, 202, 208, 220, 226, 238, 244, 256, 262, 274,280, 292, 298, 310, 316, 328, 334, 346, 352, 364, and 370; and alightchain variable region antibody sequence having a CDR1 sequence selectedfrom the group consisting of SEQ ID NOs: 5, 11, 23, 29, 41, 47, 59, 65,77, 83, 95, 101, 113, 119, 131, 137, 149, 155, 167, 173, 185, 191, 203,209, 221, 227, 239, 245, 257, 263, 275, 281, 293, 299, 311, 317, 329,335, 347, 353, 365, and 371; a CDR2 sequence selected from the groupconsisting of SEQ ID NOs: 6, 12, 24, 30, 42, 48, 60, 66, 78, 84, 96,102, 114, 120, 132, 138, 150, 156, 168, 174, 186, 192, 204, 210, 222,228, 240, 246, 258, 264, 276, 282, 294, 300, 312, 318, 330, 336, 348,354, 366, and 372; and/or a CDR3 sequence selected from the groupconsisting of SEQ ID NOs: 7, 13, 25, 31, 43, 49, 61, 67, 79, 85, 97,103, 115, 121, 133, 139, 151, 157, 169, 175, 187, 193, 205, 211, 223,229, 241, 247, 259, 265, 277, 283, 295, 301, 313, 319, 331, 337, 349,355, 367, and 373; altering at least one amino acid residue within theheavy chain variable region antibody sequence and/or the light chainvariable region antibody sequence to create at least one alteredantibody sequence; and expressing the altered antibody sequence as aprotein. The altered antibody sequence can also be prepared by screeningantibody libraries having fixed CDR3 sequences or minimal essentialbinding determinants as described in US20050255552 and diversity on CDR1and CDR2 sequences. The screening can be performed according to anyscreening technology appropriate for screening antibodies from antibodylibraries, such as phage display technology.

Standard molecular biology techniques can be used to prepare and expressthe altered antibody sequence. The antibody encoded by the alteredantibody sequence(s) is one that retains one, some or all of thefunctional properties of the HER3-binding antibodies described herein,which functional properties include, but are not limited to,specifically binding to human and/or cynomologus HER3; the antibodybinds to HER3 and neutralizes HER3 biological activity by inhibiting theHER signaling activity in a phospho-HER assay.

The functional properties of the altered antibodies can be assessedusing standard assays available in the art and/or described herein, suchas those set forth in the Examples (e.g., ELISAs).

In certain embodiments of the methods of engineering antibodies of theinvention, mutations can be introduced randomly or selectively along allor part of an HER3-binding antibody coding sequence and the resultingmodified HER3-binding antibodies can be screened for binding activityand/or other functional properties as described herein. Mutationalmethods have been described in the art. For example, PCT Publication WO02/092780 by Short describes methods for creating and screening antibodymutations using saturation mutagenesis, synthetic ligation assembly, ora combination thereof. Alternatively, PCT Publication WO 03/074679 byLazar et al. describes methods of using computational screening methodsto optimize physiochemical properties of antibodies.

Characterization of the Antibodies of the Invention

The antibodies of the invention can be characterized by variousfunctional assays. For example, they can be characterized by theirability to neutralize biological activity by inhibiting HER signaling ina phospho-HER assay as described herein, their affinity to a HER3protein (e.g., human and/or cynomologus HER3), the epitope binning,their resistance to proteolysis, and their ability to block HER3downstream signaling. Various methods can be used to measureHER3-mediated signaling. For example, the HER signaling pathway can bemonitored by (i) measurement of phospho-HER3; (ii) measurement ofphosphorylation of HER3 or other downstream signaling proteins (e.g.Akt), (iii) ligand blocking assays as described herein, (iv) heterodimerformation, (v) HER3 dependent gene expression signature, (vi) receptorinternalization, and (vii) HER3 driven cell phenotypes (e.g.proliferation).

The ability of an antibody to bind to HER3 can be detected by labellingthe antibody of interest directly, or the antibody may be unlabeled andbinding detected indirectly using various sandwich assay formats knownin the art.

In some embodiments, the HER3-binding antibodies of the invention blockor compete with binding of a reference HER3-binding antibody to a HER3polypeptide or protein. These can be fully human HER3-binding antibodiesdescribed above. They can also be other mouse, chimeric or humanizedHER3-binding antibodies which bind to the same epitope as the referenceantibody. The capacity to block or compete with the reference antibodybinding indicates that a HER3-binding antibody under test binds to thesame or similar epitope as that defined by the reference antibody, or toan epitope which is sufficiently proximal to the epitope bound by thereference HER3-binding antibody. Such antibodies are especially likelyto share the advantageous properties identified for the referenceantibody. The capacity to block or compete with the reference antibodymay be determined by, e.g., a competition binding assay. With acompetition binding assay, the antibody under test is examined forability to inhibit specific binding of the reference antibody to acommon antigen, such as a HER3 polypeptide or protein. A test antibodycompetes with the reference antibody for specific binding to the antigenif an excess of the test antibody substantially inhibits binding of thereference antibody. Substantial inhibition means that the test antibodyreduces specific binding of the reference antibody usually by at least10%, 25%, 50%, 75%, or 90%.

There are a number of known competition binding assays that can be usedto assess competition of a HER3-binding antibody with the referenceHER3-binding antibody for binding to a HER3 protein. These include,e.g., solid phase direct or indirect radioimmunoassay (RIA), solid phasedirect or indirect enzyme immunoassay (EIA), sandwich competition assay(see Stahli et al., (1983) Methods in Enzymology 9:242-253); solid phasedirect biotin-avidin EIA (see Kirkland et al., (1986) J. Immunol.137:3614-3619); solid phase direct labeled assay, solid phase directlabeled sandwich assay (see Harlow & Lane, supra); solid phase directlabel RIA using I-125 label (see Morel et al., (1988) Molec. Immunol.25:7-15); solid phase direct biotin-avidin EIA (Cheung et al., (1990)Virology 176:546-552); and direct labeled RIA (Moldenhauer et al.,(1990) Scand. J. Immunol. 32:77-82). Typically, such an assay involvesthe use of purified antigen bound to a solid surface or cells bearingeither of these, an unlabeled test HER3-binding antibody and a labelledreference antibody. Competitive inhibition is measured by determiningthe amount of label bound to the solid surface or cells in the presenceof the test antibody. Usually the test antibody is present in excess.Antibodies identified by competition assay (competing antibodies)include antibodies binding to the same epitope as the reference antibodyand antibodies binding to an adjacent epitope sufficiently proximal tothe epitope bound by the reference antibody for steric hindrance tooccur.

To determine if the selected HER3-binding monoclonal antibodies bind tounique epitopes, each antibody can be biotinylated using commerciallyavailable reagents (e.g., reagents from Pierce, Rockford, Ill.).Competition studies using unlabeled monoclonal antibodies andbiotinylated monoclonal antibodies can be performed using a HER3polypeptide coated-ELISA plates. Biotinylated MAb binding can bedetected with a strep-avidin-alkaline phosphatase probe. To determinethe isotype of a purified HER3-binding antibody, isotype ELISAs can beperformed. For example, wells of microtiter plates can be coated with 1μg/ml of anti-human IgG overnight at 4° C. After blocking with 1% BSA,the plates are reacted with 1 μg/ml or less of the monoclonalHER3-binding antibody or purified isotype controls, at ambienttemperature for one to two hours. The wells can then be reacted witheither human IgG1 or human IgM-specific alkaline phosphatase-conjugatedprobes. Plates are then developed and analyzed so that the isotype ofthe purified antibody can be determined.

To demonstrate binding of monoclonal HER3-binding antibodies to livecells expressing a HER3 polypeptide, flow cytometry can be used.Briefly, cell lines expressing HER3 (grown under standard growthconditions) can be mixed with various concentrations of a HER3-bindingantibody in PBS containing 0.1% BSA and 10% fetal calf serum, andincubated at 4° C. for 1 hour. After washing, the cells are reacted withFluorescein-labeled anti-human IgG antibody under the same conditions asthe primary antibody staining. The samples can be analyzed by FACScaninstrument using light and side scatter properties to gate on singlecells. An alternative assay using fluorescence microscopy may be used(in addition to or instead of) the flow cytometry assay. Cells can bestained exactly as described above and examined by fluorescencemicroscopy. This method allows visualization of individual cells, butmay have diminished sensitivity depending on the density of the antigen.

HER3-binding antibodies of the invention can be further tested forreactivity with a HER3 polypeptide or antigenic fragment by Westernblotting. Briefly, purified HER3 polypeptides or fusion proteins, orcell extracts from cells expressing HER3 can be prepared and subjectedto sodium dodecyl sulfate polyacrylamide gel electrophoresis. Afterelectrophoresis, the separated antigens are transferred tonitrocellulose membranes, blocked with 10% fetal calf serum, and probedwith the monoclonal antibodies to be tested. Human IgG binding can bedetected using anti-human IgG alkaline phosphatase and developed withBCIP/NBT substrate tablets (Sigma Chem. Co., St. Louis, Mo.).

A number of readouts can be used to assess the efficacy, andspecificity, of HER3 antibodies in cell-based assays of ligand-inducedheterodimer formation. Activity can be assessed by one or more of thefollowing:

(i) Inhibition of ligand-induced heterodimerisation of HER2 with otherEGF family members in a target cell line, for example MCF-7 breastcancer cells. Immunoprecipitation of HER2 complexes from cell lysatescan be performed with a receptor-specific antibody, and theabsence/presence of other EGF receptors and their biologically relevantligands within the complex can be analysed followingelectrophoresis/Western transfer by probing with antibodies to other EGFreceptors.

(ii) Inhibition of the activation of signaling pathways byligand-activated heterodimers. Association with HER3 appears key forother members of the EGF family of receptors to elicit maximal cellularresponse following ligand binding. In the case of the kinase-defectiveHER3, HER2 provides a functional tyrosine kinase domain to enablesignaling to occur following binding of growth factor ligands. Thus,cells co-expressing HER2 and HER3 can be treated with ligand, forexample heregulin, in the absence and presence of inhibitor and theeffect on HER3 tyrosine phosphorylation monitored by a number of waysincluding immunoprecipitation of HER3 from treated cell lysates andsubsequent Western blotting using anti-phosphotyrosine antibodies (seeAgus op. cit. for details). Alternatively, a high-throughput assay canbe developed by trapping HER3 from solubilized lysates onto the wells ofa 96-well plate coated with an anti-HER3 receptor antibody, and thelevel of tyrosine phosphorylation measured using, for example,europium-labelled anti-phosphotyrosine antibodies, as embodied byWaddleton et al., (2002) Anal. Biochem. 309:150-157.

In a broader extension of this approach, effector molecules known to beactivated downstream of activated receptor heterodimers, such asmitogen-activated protein kinases (MAPK) and Akt, may be analyseddirectly, by immunoprecipitation from treated lysates and blotting withantibodies that detect the activated forms of these proteins, or byanalysing the ability of these proteins to modify/activate specificsubstrates.

(iii) Inhibition of ligand-induced cellular proliferation. A variety ofcell lines are known to co-express combinations of ErbB receptors, forexample many breast and prostate cancer cell lines. Assays may beperformed in 24/48/96-well formats with the readout based around DNAsynthesis (tritiated thymidine incorporation), increase in cell number(crystal violet staining) etc.

A number of readouts can be used to assess the efficacy, andspecificity, of HER3 antibodies in cell-based assays ofligand-independent homo- and heterodimer formation. For example, HER2overexpression triggers ligand-independent activation of the kinasedomain as a result of spontaneous dimer formation. Over expressed HER2generates either homo- or heterodimers with other HER molecules such asHER1, HER3 and HER4.

Ability of antibodies or fragments thereof to block in vivo growth oftumour xenografts of human tumour cell lines whose tumorigenic phenotypeis known to be at least partly dependent on ligand activation of HER3heterodimer cell signaling e.g. BxPC3 pancreatic cancer cells etc. Thiscan be assessed in immunocompromised mice either alone or in combinationwith an appropriate cytotoxic agent for the cell line in question.Examples of functional assays are also described in the Example sectionbelow.

Prophylactic and Therapeutic Uses

The present invention provides methods of treating a disease or disorderassociated with the HER3 signaling pathway by administering to a subjectin need thereof an effective amount of the antibodies of the invention.In a specific embodiment, the present invention provides a method oftreating or preventing cancers (e.g., breast cancer, colorectal cancer,lung cancer, multiple myeloma, ovarian cancer, liver cancer, gastriccancer, pancreatic cancer, acute myeloid leukemia, chronic myeloidleukemia, osteosarcoma, squamous cell carcinoma, peripheral nerve sheathtumors, schwannoma, head and neck cancer, bladder cancer, esophagealcancer, Barretts esophageal cancer, glioblastoma, clear cell sarcoma ofsoft tissue, malignant mesothelioma, neurofibromatosis, renal cancer,melanoma, prostate cancer, benign prostatic hyperplasia (BPH),gynacomastica, and endometriosis) by administering to a subject in needthereof an effective amount of the antibodies of the invention. In someembodiments, the present invention provides methods of treating orpreventing cancers associated with a HER signaling pathway byadministering to a subject in need thereof an effective amount of theantibodies of the invention.

In a specific embodiment, the present invention provides methods oftreating cancers associated with a HER signaling pathway that include,but are not limited to breast cancer, colorectal cancer, lung cancer,multiple myeloma, ovarian cancer, liver cancer, gastric cancer,pancreatic cancer, acute myeloid leukemia, chronic myeloid leukemia,osteosarcoma, squamous cell carcinoma, peripheral nerve sheath tumorsschwannoma, head and neck cancer, bladder cancer, esophageal cancer,Barretts esophageal cancer, glioblastoma, clear cell sarcoma of softtissue, malignant mesothelioma, neurofibromatosis, renal cancer,melanoma, prostate cancer, benign prostatic hyperplasia (BPH),gynacomastica, and endometriosis.

In one embodiment, the invention pertains to treating esophageal cancerusing the HER3 antibodies or fragments thereof.

In one embodiment, the invention pertains to treating benign prostatichyperplasia (BPH) using the HER3 antibodies or fragments thereof. BHP isa common disease in aging males that is characterized by anon-neoplastic enlargement of the prostate leading to pressure on theurethra and resulting in urination and bladder problems (Mahapokail W,van Sluijs F J & Schalken J A. 2000 Prostate Cancer and ProstaticDiseases 3, 28-33). Anatomic or microscopic evidence of BPH is presentat autopsy in approximately 55% of men aged 60-70 years. Transurethralresection of the prostate has been the treatment of choice for manyyears. Consequently, BPH is one of the most common reasons for surgicalintervention among elderly men. Less invasive methods of treatmentinclude:

(i) Alpha 1-blockers (doxazosin, prazosin, tamsulosin, terazosin, andalfuzosin) are a class of medications also used to treat high bloodpressure. These medications relax the muscles of the bladder neck andprostate thus allowing easier urination.

(ii) Finasteride and dutasteride lower androgen levels thus reducing thesize of the prostate gland, increasing urine flow rate, and decreasingsymptoms of BPH. It may take 3 to 6 months before improvement insymptoms is observed. Potential side effects related to the use offinasteride and dutasteride include decreased sex drive and impotence

The results in the Experiments section demonstrate for the first timethat BPH is a neuregulin-dependent indication. The finding also showthat MOR0703 significantly reduced prostate size in sexually mature ratswithout affecting hormone levels suggests that MORI0703 could be usefulfor the treatment of BPH.

To further test MOR10703 and other HER3 antibodies as therapeutics forBPH, primary human BPH surgical specimens can be transplanted intoathymic mice or rats and effect of the Her3 antibodies studied usingmodels (Otto U et al. Urol Int 1992; 48: 167-170). Alternatively,aspects of BPH can be induced in castrated dogs via the long-termadministration of 5α-Androstane-3u, 170-diol plus estradiol and the HER3antibodies tested in these canine models (Walsh P C, Wilson J D. J ClinInvest 1976; 57: 1093-1097).

In one embodiment, the invention pertains to treating gynecomastia usingthe HER3 antibodies or fragments thereof. The physical manifestation ofgynecomastia is breast enlargement in males, it normally occurs in bothbreasts but can sometimes be in one only, known as asymmetrical orunilateral gynecosmastia. Gynecomastia is commonly caused by:

(i) Elevated estrogen levels resulting in the ratio of testosterone tooestrogen becoming unbalanced.

(ii) Androgen antagonists or anti-androgens used in the treatment ofprostate cancer or BPH. These drugs suppress testosterone but bysuppressing testosterone, oestrogen begins to rise.

Although a number of experimental drugs are currently being evaluatedthere are currently no approved treatments for gynecomastia.Consequently gynecomastia is treated via surgical removal of the breasttissue. The observation that MORI0703 induced irreversible atrophy ofthe male mammary gland indicates that it may be of benefit in thetreatment of gynecomastia.

To further test MOR10703 and other HER3 antibodies for treating humangynecomastia, transgenic mouse models of gynecomastia can be used. Thesemodels have been developed by expressing human aromatase in the mousemammary gland and recapitulate many aspects of human gynecomastia (Li etal., Endocrinology 2002; 143:4074-4083; Tekmal et al., Cancer Res 1996;56:3180-318).

In one embodiment, the invention pertains to treating gynecomastia usingthe HER3 antibodies or fragments thereof. MOR10703 and other HER3antibodies can also be examined for treating endometriosis, agynecological condition in which cells from the lining of the uterus(endometrium) appear and flourish outside the uterine cavity. The main,but not universal, symptom of endometriosis is pelvic pain in variousmanifestations. Although the underlying causes of endometriosis are notwell characterized is thought to be dependent on the presence ofestrogen. Since MOR10703 induced endometrial atrophy in female miceresulting in a decrease in uterus weight, it may be used to treatendometriosis. To further study the effects of MORI0703 and other HER3antibodies on endometriosis, human endometrium in the proliferativephase can be implanted into the peritoneal cavity of normal cycling andovariectomized athymic mice or cycling non-obese diabetic (NOD)-severecombined immuno-deficiency (SCID) mice, and these SCID mice used asmodels (Grummer et al., 2001. Human Reproduction; 16; 1736-1743).

HER3 antibodies can also be used to treat or prevent other disordersassociated with aberrant or defective HER signaling, including but arenot limited to respiratory diseases, osteoporosis, osteoarthritis,polycystic kidney disease, diabetes, schizophrenia, vascular disease,cardiac disease, non-oncogenic proliferative diseases, fibrosis, andneurodegenerative diseases such as Alzheimer's disease.

Suitable agents for combination treatment with HER3-binding antibodiesinclude standard of care agents known in the art that are able tomodulate the ErbB signaling pathway. Suitable examples of standard ofcare agents for HER2 include, but are not limited to Herceptin andTykerb. Suitable examples of standard of care agents for EGFR include,but are not limited to Iressa, Tarceva, Erbitux and Vectibix. Otheragents that may be suitable for combination treatment with HER3-bindingantibodies include, but are not limited to those that modulate receptortyrosine kinases, G-protein coupled receptors, growth/survival signaltransduction pathways, nuclear hormone receptors, apoptotic pathways,cell cycle and angiogenesis.

Diagnostic Uses

In one aspect, the invention encompasses diagnostic assays fordetermining HER3 protein and/or nucleic acid expression as well as HER3protein function, in the context of a biological sample (e.g., blood,serum, cells, tissue) or from individual afflicted with cancer, or is atrisk of developing cancer.

Diagnostic assays, such as competitive assays rely on the ability of alabelled analogue (the “tracer”) to compete with the test sample analytefor a limited number of binding sites on a common binding partner. Thebinding partner generally is insolubilized before or after thecompetition and then the tracer and analyte bound to the binding partnerare separated from the unbound tracer and analyte. This separation isaccomplished by decanting (where the binding partner waspreinsolubilized) or by centrifuging (where the binding partner wasprecipitated after the competitive reaction). The amount of test sampleanalyte is inversely proportional to the amount of bound tracer asmeasured by the amount of marker substance. Dose-response curves withknown amounts of analyte are prepared and compared with the test resultsin order to quantitatively determine the amount of analyte present inthe test sample. These assays are called ELISA systems when enzymes areused as the detectable markers. In an assay of this form, competitivebinding between antibodies and HER3-binding antibodies results in thebound HER3 protein, preferably the HER3 epitopes of the invention, beinga measure of antibodies in the serum sample, most particularly,neutralizing antibodies in the serum sample.

A significant advantage of the assay is that measurement is made ofneutralizing antibodies directly (i.e., those which interfere withbinding of HER3 protein, specifically, epitopes). Such an assay,particularly in the form of an ELISA test has considerable applicationsin the clinical environment and in routine blood screening.

Another aspect of the invention provides methods for determining HER3nucleic acid expression or HER3 protein activity in an individual tothereby select appropriate therapeutic or prophylactic agents for thatindividual (referred to herein as “pharmacogenomics”). Pharmacogenomicsallows for the selection of agents (e.g., drugs) for therapeutic orprophylactic treatment of an individual based on the genotype of theindividual (e.g., the genotype of the individual examined to determinethe ability of the individual to respond to a particular agent.)

Yet another aspect of the invention pertains to monitoring the influenceof agents (e.g., drugs) on the expression or activity of HER3 protein inclinical trials.

Pharmaceutical Compositions

To prepare pharmaceutical or sterile compositions including aHER3-binding antibodies (intact or binding fragments), the HER3-bindingantibodies (intact or binding fragments) is mixed with apharmaceutically acceptable carrier or excipient. The compositions canadditionally contain one or more other therapeutic agents that aresuitable for treating or preventing cancer (breast cancer, colorectalcancer, lung cancer, multiple myeloma, ovarian cancer, liver cancer,gastric cancer, pancreatic cancer, acute myeloid leukemia, chronicmyeloid leukemia, osteosarcoma, squamous cell carcinoma, peripheralnerve sheath tumors schwannoma, head and neck cancer, bladder cancer,esophageal cancer, Barretts esophageal cancer, glioblastoma, clear cellsarcoma of soft tissue, malignant mesothelioma, neurofibromatosis, renalcancer, melanoma, prostate cancer, benign prostatic hyperplasia (BPH),gynacomastica, and endometriosis).

Formulations of therapeutic and diagnostic agents can be prepared bymixing with physiologically acceptable carriers, excipients, orstabilizers in the form of, e.g., lyophilized powders, slurries, aqueoussolutions, lotions, or suspensions (see, e.g., Hardman et al., (2001)Goodman and Gilman's The Pharmacological Basis of Therapeutics,McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: The Science andPractice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y.;Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: ParenteralMedications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990)Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, etal. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, MarcelDekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety,Marcel Dekker, Inc., New York, N.Y.).

Selecting an administration regimen for a therapeutic depends on severalfactors, including the serum or tissue turnover rate of the entity, thelevel of symptoms, the immunogenicity of the entity, and theaccessibility of the target cells in the biological matrix. In certainembodiments, an administration regimen maximizes the amount oftherapeutic delivered to the patient consistent with an acceptable levelof side effects. Accordingly, the amount of biologic delivered dependsin part on the particular entity and the severity of the condition beingtreated. Guidance in selecting appropriate doses of antibodies,cytokines, and small molecules are available (see, e.g., Wawrzynczak(1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK;Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis,Marcel Dekker, New York, N.Y.; Bach (ed.) (1993) Monoclonal Antibodiesand Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York,N.Y.; Baert et al., (2003) New Engl. J. Med. 348:601-608; Milgrom etal., (1999) New Engl. J. Med. 341:1966-1973; Slamon et al., (2001) NewEngl. J. Med. 344:783-792; Beniaminovitz et al., (2000) New Engl. J.Med. 342:613-619; Ghosh et al., (2003) New Engl. J. Med. 348:24-32;Lipsky et al., (2000) New Engl. J. Med. 343:1594-1602).

Determination of the appropriate dose is made by the clinician, e.g.,using parameters or factors known or suspected in the art to affecttreatment or predicted to affect treatment. Generally, the dose beginswith an amount somewhat less than the optimum dose and it is increasedby small increments thereafter until the desired or optimum effect isachieved relative to any negative side effects. Important diagnosticmeasures include those of symptoms of, e.g., the inflammation or levelof inflammatory cytokines produced.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors known in the medical arts.

Compositions comprising antibodies or fragments thereof of the inventioncan be provided by continuous infusion, or by doses at intervals of,e.g., one day, one week, or 1-7 times per week. Doses may be providedintravenously, subcutaneously, topically, orally, nasally, rectally,intramuscular, intracerebrally, or by inhalation. A specific doseprotocol is one involving the maximal dose or dose frequency that avoidssignificant undesirable side effects. A total weekly dose may be atleast 0.05 μg/kg body weight, at least 0.2 μg/kg, at least 0.5 μg/kg, atleast 1 μg/kg, at least 10 μg/kg, at least 100 μg/kg, at least 0.2mg/kg, at least 1.0 mg/kg, at least 2.0 mg/kg, at least 10 mg/kg, atleast 25 mg/kg, or at least 50 mg/kg (see, e.g., Yang et al., (2003) NewEngl. J. Med. 349:427-434; Herold et al., (2002) New Engl. J. Med.346:1692-1698; Liu et al., (1999) J. Neurol. Neurosurg. Psych.67:451-456; Portielji et al., (2003) Cancer Immunol. Immunother.52:133-144). The desired dose of antibodies or fragments thereof isabout the same as for an antibody or polypeptide, on a moles/kg bodyweight basis. The desired plasma concentration of the antibodies orfragments thereof is about, on a moles/kg body weight basis. The dosemay be at least 15 μg at least 20 μg, at least 25 μg, at least 30 μg, atleast 35 μg, at least 40 μg, at least 45 μg, at least 50 μg, at least 55μg, at least 60 μg, at least 65 μg, at least 70 μg, at least 75 μg, atleast 80 μg, at least 85 μg, at least 90 μg, at least 95 μg, or at least100 μg. The doses administered to a subject may number at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, or 12, or more.

For antibodies or fragments thereof of the invention, the dosageadministered to a patient may be 0.0001 mg/kg to 100 mg/kg of thepatient's body weight. The dosage may be between 0.0001 mg/kg and 20mg/kg, 0.0001 mg/kg and 10 mg/kg, 0.0001 mg/kg and 5 mg/kg, 0.0001 and 2mg/kg, 0.0001 and 1 mg/kg, 0.0001 mg/kg and 0.75 mg/kg, 0.0001 mg/kg and0.5 mg/kg, 0.0001 mg/kg to 0.25 mg/kg, 0.0001 to 0.15 mg/kg, 0.0001 to0.10 mg/kg, 0.001 to 0.5 mg/kg, 0.01 to 0.25 mg/kg or 0.01 to 0.10 mg/kgof the patient's body weight.

The dosage of the antibodies or fragments thereof of the invention maybe calculated using the patient's weight in kilograms (kg) multiplied bythe dose to be administered in mg/kg. The dosage of the antibodies orfragments thereof of the invention may be 150 μg/kg or less, 125 μg/kgor less, 100 μg/kg or less, 95 μg/kg or less, 90 μg/kg or less, 85 g/kgor less, 80 g/kg or less, 75 μg/kg or less, 70 μg/kg or less, 65 μg/kgor less, 60 g/kg or less, 55 g/kg or less, 50 μg/kg or less, 45 μg/kg orless, 40 μg/kg or less, 35 μg/kg or less, 30 g/kg or less, 25 μg/kg orless, 20 μg/kg or less, 15 μg/kg or less, 10 g/kg or less, 5 g/kg orless, 2.5 g/kg or less, 2 g/kg or less, 1.5 μg/kg or less, 1 g/kg orless, 0.5 μg/kg or less, or 0.5 g/kg or less of a patient's body weight.

Unit dose of the antibodies or fragments thereof of the invention may be0.1 mg to 20 mg, 0.1 mg to 15 mg, 0.1 mg to 12 mg, 0.1 mg to 10 mg, 0.1mg to 8 mg, 0.1 mg to 7 mg, 0.1 mg to 5 mg, 0.1 to 2.5 mg, 0.25 mg to 20mg, 0.25 to 15 mg, 0.25 to 12 mg, 0.25 to 10 mg, 0.25 to 8 mg, 0.25 mgto 7 m g, 0.25 mg to 5 mg, 0.5 mg to 2.5 mg, 1 mg to 20 mg, 1 mg to 15mg, 1 mg to 12 mg, 1 mg to 10 mg, 1 mg to 8 mg, 1 mg to 7 mg, 1 mg to 5mg, or 1 mg to 2.5 mg.

The dosage of the antibodies or fragments thereof of the invention mayachieve a serum titer of at least 0.1 μg/ml, at least 0.5 μg/ml, atleast 1 μg/ml, at least 2 μg/ml, at least 5 g/ml, at least 6 g/ml, atleast 10 μg/ml, at least 15 μg/ml, at least 20 g/ml, at least 25 g/ml,at least 50 μg/ml, at least 100 μg/ml, at least 125 μg/ml, at least 150μg/ml, at least 175 μg/ml, at least 200 μg/ml, at least 225 μg/ml, atleast 250 g/ml, at least 275 μg/ml, at least 300 g/ml, at least 325μg/ml, at least 350 μg/ml, at least 375 μg/ml, or at least 400 μg/ml ina subject. Alternatively, the dosage of the antibodies or fragmentsthereof of the invention may achieve a serum titer of at least 0.1μg/ml, at least 0.5 μg/ml, at least 1 μg/ml, at least, 2 μg/ml, at least5 μg/ml, at least 6 g/ml, at least 10 μg/ml, at least 15 μg/ml, at least20 .mu.g/ml, at least 25 g/ml, at least 50 μg/ml, at least 100 μg/ml, atleast 125 μg/ml, at least 150 μg/ml, at least 175 μg/ml, at least 200μg/ml, at least 225 μg/ml, at least 250 μg/ml, at least 275 μg/ml, atleast 300 μg/ml, at least 325 μg/ml, at least 350 μg/ml, at least 375μg/ml, or at least 400 μg/ml in the subject.

Doses of antibodies or fragments thereof of the invention may berepeated and the administrations may be separated by at least 1 day, 2days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75days, 3 months, or at least 6 months.

An effective amount for a particular patient may vary depending onfactors such as the condition being treated, the overall health of thepatient, the method route and dose of administration and the severity ofside affects (see, e.g., Maynard et al., (1996) A Handbook of SOPs forGood Clinical Practice, Interpharm Press, Boca Raton, Fla.; Dent (2001)Good Laboratory and Good Clinical Practice, Urch Publ., London, UK).

The route of administration may be by, e.g., topical or cutaneousapplication, injection or infusion by intravenous, intraperitoneal,intracerebral, intramuscular, intraocular, intraarterial,intracerebrospinal, intralesional, or by sustained release systems or animplant (see, e.g., Sidman et al., (1983) Biopolymers 22:547-556; Langeret al., (1981) J. Biomed. Mater. Res. 15:167-277; Langer (1982) Chem.Tech. 12:98-105; Epstein et al., (1985) Proc. Natl. Acad. Sci. USA82:3688-3692; Hwang et al., (1980) Proc. Natl. Acad. Sci. USA77:4030-4034; U.S. Pat. Nos. 6,350,466 and 6,316,024). Where necessary,the composition may also include a solubilizing agent and a localanesthetic such as lidocaine to ease pain at the site of the injection.In addition, pulmonary administration can also be employed, e.g., by useof an inhaler or nebulizer, and formulation with an aerosolizing agent.See, e.g., U.S. Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272,5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT Publication Nos.WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903,each of which is incorporated herein by reference their entirety.

A composition of the present invention may also be administered via oneor more routes of administration using one or more of a variety ofmethods known in the art. As will be appreciated by the skilled artisan,the route and/or mode of administration will vary depending upon thedesired results. Selected routes of administration for antibodies orfragments thereof of the invention include intravenous, intramuscular,intradermal, intraperitoneal, subcutaneous, spinal or other parenteralroutes of administration, for example by injection or infusion.Parenteral administration may represent modes of administration otherthan enteral and topical administration, usually by injection, andincludes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion. Alternatively, a composition of theinvention can be administered via a non-parenteral route, such as atopical, epidermal or mucosal route of administration, for example,intranasally, orally, vaginally, rectally, sublingually or topically. Inone embodiment, the antibodies or fragments thereof of the invention isadministered by infusion. In another embodiment, the multispecificepitope binding protein of the invention is administered subcutaneously.

If the antibodies or fragments thereof of the invention are administeredin a controlled release or sustained release system, a pump may be usedto achieve controlled or sustained release (see Langer, supra; Sefton,(1987) CRC Crit. Ref Biomed. Eng. 14:20; Buchwald et al., (1980),Surgery 88:507; Saudek et al., (1989) N. Engl. J. Med. 321:574).Polymeric materials can be used to achieve controlled or sustainedrelease of the therapies of the invention (see e.g., MedicalApplications of Controlled Release, Langer and Wise (eds.), CRC Pres.,Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug ProductDesign and Performance, Smolen and Ball (eds.), Wiley, New York (1984);Ranger and Peppas, (1983) J. Macromol. Sci. Rev. Macromol. Chem. 23:61;see also Levy et al., (1985) Science 228:190; During et al., (1989) Ann.Neurol. 25:351; Howard et al., (1989) J. Neurosurg. 7 1:105); U.S. Pat.Nos. 5,679,377; 5,916,597; 5,912,015; 5,989,463; 5,128,326; PCTPublication No. WO 99/15154; and PCT Publication No. WO 99/20253.Examples of polymers used in sustained release formulations include, butare not limited to, poly(2-hydroxy ethyl methacrylate), poly(methylmethacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate),poly(methacrylic acid), polyglycolides (PLG), polyanhydrides,poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide,poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides)(PLGA), and polyorthoesters. In one embodiment, the polymer used in asustained release formulation is inert, free of leachable impurities,stable on storage, sterile, and biodegradable. A controlled or sustainedrelease system can be placed in proximity of the prophylactic ortherapeutic target, thus requiring only a fraction of the systemic dose(see, e.g., Goodson, in Medical Applications of Controlled Release,supra, vol. 2, pp. 115-138 (1984)).

Controlled release systems are discussed in the review by Langer,(1990), Science 249:1527-1533). Any technique known to one of skill inthe art can be used to produce sustained release formulations comprisingone or more antibodies or fragments thereof of the invention. See, e.g.,U.S. Pat. No. 4,526,938, PCT publication WO 91/05548, PCT publication WO96/20698, Ning et al., (1996), Radiotherapy & Oncology 39:179-189, Songet al., (1995) PDA Journal of Pharmaceutical Science & Technology50:372-397, Cleek et al., (1997) Pro. Int'l. Symp. Control. Rel. Bioact.Mater. 24:853-854, and Lam et al., (1997) Proc. Int'l. Symp. ControlRel. Bioact. Mater. 24:759-760, each of which is incorporated herein byreference in their entirety.

If the antibodies or fragments thereof of the invention are administeredtopically, they can be formulated in the form of an ointment, cream,transdermal patch, lotion, gel, shampoo, spray, aerosol, solution,emulsion, or other form well-known to one of skill in the art. See,e.g., Remington's Pharmaceutical Sciences and Introduction toPharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa.(1995). For non-sprayable topical dosage forms, viscous to semi-solid orsolid forms comprising a carrier or one or more excipients compatiblewith topical application and having a dynamic viscosity, in someinstances, greater than water are typically employed. Suitableformulations include, without limitation, solutions, suspensions,emulsions, creams, ointments, powders, liniments, salves, and the like,which are, if desired, sterilized or mixed with auxiliary agents (e.g.,preservatives, stabilizers, wetting agents, buffers, or salts) forinfluencing various properties, such as, for example, osmotic pressure.Other suitable topical dosage forms include sprayable aerosolpreparations wherein the active ingredient, in some instances, incombination with a solid or liquid inert carrier, is packaged in amixture with a pressurized volatile (e.g., a gaseous propellant, such asfreon) or in a squeeze bottle. Moisturizers or humectants can also beadded to pharmaceutical compositions and dosage forms if desired.Examples of such additional ingredients are well-known in the art.

If the compositions comprising antibodies or fragments thereof areadministered intranasally, it can be formulated in an aerosol form,spray, mist or in the form of drops. In particular, prophylactic ortherapeutic agents for use according to the present invention can beconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebuliser, with the use of a suitable propellant(e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridges(composed of, e.g., gelatin) for use in an inhaler or insufflator may beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

Methods for co-administration or treatment with a second therapeuticagent, e.g., a cytokine, steroid, chemotherapeutic agent, antibiotic, orradiation, are known in the art (see, e.g., Hardman et al., (eds.)(2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics,10.sup.th ed., McGraw-Hill, New York, N.Y.; Poole and Peterson (eds.)(2001) Pharmacotherapeutics for Advanced Practice: A Practical Approach,Lippincott, Williams & Wilkins, Phila., Pa.; Chabner and Longo (eds.)(2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams &Wilkins, Phila., Pa.). An effective amount of therapeutic may decreasethe symptoms by at least 10%; by at least 20%; at least about 30%; atleast 40%, or at least 50%.

Additional therapies (e.g., prophylactic or therapeutic agents), whichcan be administered in combination with the antibodies or fragmentsthereof of the invention may be administered less than 5 minutes apart,less than 30 minutes apart, 1 hour apart, at about 1 hour apart, atabout 1 to about 2 hours apart, at about 2 hours to about 3 hours apart,at about 3 hours to about 4 hours apart, at about 4 hours to about 5hours apart, at about 5 hours to about 6 hours apart, at about 6 hoursto about 7 hours apart, at about 7 hours to about 8 hours apart, atabout 8 hours to about 9 hours apart, at about 9 hours to about 10 hoursapart, at about 10 hours to about 11 hours apart, at about 11 hours toabout 12 hours apart, at about 12 hours to 18 hours apart, 18 hours to24 hours apart, 24 hours to 36 hours apart, 36 hours to 48 hours apart,48 hours to 52 hours apart, 52 hours to 60 hours apart, 60 hours to 72hours apart, 72 hours to 84 hours apart, 84 hours to 96 hours apart, or96 hours to 120 hours apart from the antibodies or fragments thereof ofthe invention. The two or more therapies may be administered within onesame patient visit.

The antibodies or fragments thereof of the invention and the othertherapies may be cyclically administered. Cycling therapy involves theadministration of a first therapy (e.g., a first prophylactic ortherapeutic agent) for a period of time, followed by the administrationof a second therapy (e.g., a second prophylactic or therapeutic agent)for a period of time, optionally, followed by the administration of athird therapy (e.g., prophylactic or therapeutic agent) for a period oftime and so forth, and repeating this sequential administration, i.e.,the cycle in order to reduce the development of resistance to one of thetherapies, to avoid or reduce the side effects of one of the therapies,and/or to improve the efficacy of the therapies.

In certain embodiments, the antibodies or fragments thereof of theinvention can be formulated to ensure proper distribution in vivo. Forexample, the blood-brain barrier (BBB) excludes many highly hydrophiliccompounds. To ensure that the therapeutic compounds of the inventioncross the BBB (if desired), they can be formulated, for example, inliposomes. For methods of manufacturing liposomes, see, e.g., U.S. Pat.Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise oneor more moieties which are selectively transported into specific cellsor organs, thus enhance targeted drug delivery (see, e.g., Ranade,(1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties includefolate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al);mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun.153:1038); antibodies (Bloeman et al., (1995) FEBS Lett. 357:140; Owaiset al., (1995) Antimicrob. Agents Chemother. 39:180); surfactant proteinA receptor (Briscoe et al., (1995) Am. J. Physiol. 1233:134); p 120(Schreier et al, (1994) J. Biol. Chem. 269:9090); see also K. Keinanen;M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I. J. Fidler(1994) Immunomethods 4:273.

The invention provides protocols for the administration ofpharmaceutical composition comprising antibodies or fragments thereof ofthe invention alone or in combination with other therapies to a subjectin need thereof. The therapies (e.g., prophylactic or therapeuticagents) of the combination therapies of the present invention can beadministered concomitantly or sequentially to a subject. The therapy(e.g., prophylactic or therapeutic agents) of the combination therapiesof the present invention can also be cyclically administered. Cyclingtherapy involves the administration of a first therapy (e.g., a firstprophylactic or therapeutic agent) for a period of time, followed by theadministration of a second therapy (e.g., a second prophylactic ortherapeutic agent) for a period of time and repeating this sequentialadministration, i.e., the cycle, in order to reduce the development ofresistance to one of the therapies (e.g., agents) to avoid or reduce theside effects of one of the therapies (e.g., agents), and/or to improve,the efficacy of the therapies.

The therapies (e.g., prophylactic or therapeutic agents) of thecombination therapies of the invention can be administered to a subjectconcurrently. The term “concurrently” is not limited to theadministration of therapies (e.g., prophylactic or therapeutic agents)at exactly the same time, but rather it is meant that a pharmaceuticalcomposition comprising antibodies or fragments thereof of the inventionare administered to a subject in a sequence and within a time intervalsuch that the antibodies of the invention can act together with theother therapy(ies) to provide an increased benefit than if they wereadministered otherwise. For example, each therapy may be administered toa subject at the same time or sequentially in any order at differentpoints in time; however, if not administered at the same time, theyshould be administered sufficiently close in time so as to provide thedesired therapeutic or prophylactic effect. Each therapy can beadministered to a subject separately, in any appropriate form and by anysuitable route. In various embodiments, the therapies (e.g.,prophylactic or therapeutic agents) are administered to a subject lessthan 15 minutes, less than 30 minutes, less than 1 hour apart, at about1 hour apart, at about 1 hour to about 2 hours apart, at about 2 hoursto about 3 hours apart, at about 3 hours to about 4 hours apart, atabout 4 hours to about 5 hours apart, at about 5 hours to about 6 hoursapart, at about 6 hours to about 7 hours apart, at about 7 hours toabout 8 hours apart, at about 8 hours to about 9 hours apart, at about 9hours to about 10 hours apart, at about 10 hours to about 11 hoursapart, at about 11 hours to about 12 hours apart, 24 hours apart, 48hours apart, 72 hours apart, or 1 week apart. In other embodiments, twoor more therapies (e.g., prophylactic or therapeutic agents) areadministered to a within the same patient visit.

The prophylactic or therapeutic agents of the combination therapies canbe administered to a subject in the same pharmaceutical composition.Alternatively, the prophylactic or therapeutic agents of the combinationtherapies can be administered concurrently to a subject in separatepharmaceutical compositions. The prophylactic or therapeutic agents maybe administered to a subject by the same or different routes ofadministration.

The invention having been fully described, it is further illustrated bythe following examples and claims, which are illustrative and are notmeant to be further limiting.

EXAMPLES Example 1: Methods, Materials and Screening for Antibodies

(i) Cell Lines

BXPC-3, SK-Br-3, BT-474, MDA-MB-453, FaDu and MCF-7 cell lines werepurchased from ATCC and routinely maintained in growth mediasupplemented with 10% fetal bovine serum (FBS).

(ii) Generation of Recombinant Human, Cyno, Mouse and Rat HER3 Vectors

Murine HER3 extracellular domain was PCR amplified from mouse brain cDNA(Clontech) and sequence verified by comparison with Refseq NM_010153.Rat HER3 ECD was reverse transcribed from Rat-2 cell mRNA and sequenceverified by comparison with NM_017218. Cynomolgus HER3 cDNA template wasgenerated using RNA from various cyno tissues (Zyagen Laboratories), andthe RT-PCR product cloned into pCR-TOPO-XL (Invitrogen) prior tosequencing of both strands. Human HER3 was derived from a human fetalbrain cDNA library (Source) and sequence verified by comparison withNM_001982.

To generate tagged recombinant proteins, human, mouse, rat and cyno HER3was PCR amplified using Pwo Taq polymerase (Roche Diagnostics).Amplified PCR products were gel purified and cloned into a pDonR201(Invitrogen) gateway entry vector that had previously been modified toinclude an in-frame N-terminal CD33 leader sequence and a C-terminalTAG, e.g., FLAG TAG. The TAG allows purification of monomeric proteinsvia an anti-TAG monoclonal antibody. The target genes were flanked withAttB1 and AttB2 allowing recombination into Gateway adapted proprietarydestination vectors (e.g., pcDNA3.1) using the Gateway® cloningtechnology (Invitrogen). Recombination reactions were performed using aGateway LR reaction with proprietary destination vectors containing aCMV promoter to create the TAG expression vectors, although anycommercially available vector can be used.

Further recombinant HER3 proteins were generated that fused the HER3 ECDupstream of a C-terminal Factor X cleavage site and the human IgG hingeand Fc domain to create an Fc-tagged protein. To achieve this, thevarious HER3 ECD's were PCR amplified and cloned into a vector (e.g.,pcDNA3.1) modified to contain an in-frame C-terminal fusion of Factor Xsite-Hinge-hFc. The generated open reading frame was flanked with AttB1and AttB2 sites for further cloning with the Gateway® recombinantcloning technology (Invitrogen). An LR Gateway reaction was used totransfer HER3-Fc into a destination expression construct containing aCMV promoter. HER3 point mutation expression constructs were generatedusing standard site directed mutagenesis protocols and the resultantvectors sequence verified.

TABLE 8 Generation of HER3 expression vectors. HER3 amino acid numberingis based on NP_001973 (human), NP_034283 (mouse) and NP_058914 (rat).Name Description Hu HER3 CD33-[Human HER3, residues 20-640]-TAG Mu HER3CD33-[Murine HER3, residues 20-643]-TAG Rat HER3 CD33-[Rat HER3,residues 20-643]-TAG Cyno HER3 CD33-[Cyno HER3, residues 20-643]-TAGHER3 D1-2 CD33-[Human HER3, residues 20-329]-TAG HER3 D2 CD33-[HumanHER3, residues 185-329]-TAG HER3 D3-4 CD33-[Human HER3, residues330-643]-TAG HER3 D4 CD33-[Human HER3, residues 496-643]-TAG Hu HER3-Fc[Human HER3, residues 1-643]-Fc Mu HER3-Fc [Murine HER3, residues1-643]-Fc Cyno HER3-Fc [Cyno HER3, residues 1-643]-Fc Rat HER3-Fc [RatHER3, residues 1-643]-Fc HER3 D2-Fc [Human HER3 residues 207-329]-FcHER3 K267A CD33-[Human HER3, residues 20-640, K267A]-TAG HER3 L268ACD33-[Human HER3, residues 20-640, L268A]-TAG HER3 K267A/ CD33-[HumanHER3, residues 20-640, K267A/ L268A L268A]-TAG

(iii) Expression of Recombinant HER3 Proteins

The desired HER3 recombinant proteins were expressed in HEK293 derivedcell lines previously adapted to suspension culture and grown in aNovartis proprietary serum-free medium. Small scale expressionverification was undertaken in transient 6-well-plate transfectionassays on the basis of lipofection. Large-scale protein production viatransient transfection and was performed at the 10-20 L scale in theWave™ bioreactor system (Wave Biotech). DNA Polyethylenimine(Polysciences) was used as a plasmid carrier at a ratio of 1:3 (w:w).The cell culture supernatants were harvested 7-10 days post transfectionand concentrated by cross-flow filtration and diafiltration prior topurification.

(iv) Tagged Protein Purification

Recombinant tagged HER3 proteins (e.g., TAG-HER3) were purified bycollecting the cell culture supernatant and concentrating 10-fold bycross-flow filtration with a 10 kDa cut off filter (Fresenius). Ananti-TAG column was prepared by coupling an anti-TAG monoclonal antibodyto CNBr activated Sepharose 4B at a final ratio of 10 mg antibody per mLof resin. Concentrated supernatant was applied to a 35 ml anti-Tagcolumn at a flow rate of 1-2 mL/minute. After base-line washing withPBS, bound material was eluted with 100 mM glycine (pH 2.7), neutralizedand sterile filtered. Protein concentrations were determined bymeasuring the absorbance at 280 nm and converting using a theoreticalfactor of 0.66 AU/mg. The purified protein was finally characterized bySDS-PAGE, N-terminal sequencing and LC-MS.

(v) Fc Tag Purification

Concentrated cell culture supernatant was applied to a 50 ml Protein ASepharose Fast Flow column at a flow rate of 1 ml/min. After baselinewashing with PBS, the column was washed with 10 column volumes of 10 mMNaH₂PO₄/30% (v/v) Isopropanol, pH 7.3 followed by 5 column volumes ofPBS. Finally, bound material was eluted with 50 mM Citrate/140 mM NaCl(pH 2.7), neutralized and sterile filtered.

(vi) Generation of Over-Expressing Cell Lines

To generate a cell line that expresses high levels of HER3 on the cellsurface, a mammalian expression vector was constructed containing aninsert coding for a CD33 leader sequence upstream of amino acid residues20-667 of human HER3 fused in-frame to amino acid residues 669-1210 ofhuman EGFR. When expressed in mammalian cells the resultant chimericprotein contains an N-terminal HER3 extracellular and transmembranedomain and a C-terminal EGFR cytoplasmic domain. The HER3/1 vector wastransfected into CHO-S cells (Invitrogen) and stable pools generatedfollowing antibiotic selection. The resultant cell line (CHO HER3/1)expressed high levels of HER3 extracellular domain on its cell surface.

(vii) HuCAL GOLD® Pannings

For the selection of antibodies recognizing human HER3 multiple panningstrategies were employed. Therapeutic antibodies against human HER3protein were generated by selection of clones having high bindingaffinities, using as the source of antibody variant proteins acommercially available phage display library, the MorphoSys HuCAL GOLD®library. The phagemid library is based on the HuCAL® concept (Knappik etal., (2000) J Mol Biol 296:57-86) and employs the CysDisplay® technologyfor displaying the Fab on the phage surface (WO01/05950 to Lohning).

For the isolation of anti-HER3 antibodies, standard as well as RapMATpanning strategies were performed using solid phase, solution, wholecell and differential whole cell panning approaches.

(viii) Solid Phase Panning

To identify anti-HER3 antibodies a variety of solid phase panningstrategies were performed using differing recombinant HER3 proteins. Toperform each round of solid phase panning, Maxisorp plates (Nunc) werecoated with HER3 protein. Tagged proteins were either captured usingplates previously coated with anti-Fc (goat or mouse anti-human IgG,Jackson Immuno Research), anti-Tag antibody or via passive adsorption.The coated plates were washed with PBS and blocked. Coated plates werewashed twice with PBS prior to the addition of HuCAL GOLD®phage-antibodies for 2 hours at room temperature on a shaker. Boundphages were eluted were added to E. coli TG-1 and incubated for phageinfection. Subsequently infected bacteria were isolated and plated onagar plates. Colonies were scraped off the plates and phages wererescued and amplified. Each HER3 panning strategy comprised ofindividual rounds of panning and contained unique antigens, antigenconcentrations and washing stringency.

(ix) Solution Phase Panning

Each round of solution phase panning was performed using variousbiotinylated recombinant HER3 proteins in the presence or absence ofneuregulin 1-01 (R&D Systems). Proteins were biotinylated using theEZ-link sulfo-NHS-LC biotinylation kit (Pierce) according to themanufacturers instructions. 800 μl of Streptavidin linked magnetic beads(Dynabeads, Dynal) were washed once with PBS and blocked overnight withChemiblocker (Chemicon). HuCAL GOLD® phage-antibodies and theappropriate biotinylated HER3 were incubated in a reaction tube.Streptavidin magnetic beads were added for 20 minutes and were collectedwith a magnetic particle separator (Dynal). Bound phages were elutedfrom the Dynabeads by adding DTT containing buffer to each tube andadded to E. coli TG-1. Phage infection was performed in an identicalmanner to that described in solid phase panning. Each HER3 panningstrategy comprised of individual rounds of panning and contained uniqueantigens, antigen concentrations and washing stringency.

(x) Cell Based Panning

For cell pannings, HuCAL GOLD© phage-antibodies were incubated withapproximately 10⁷ cells on a rotator for 2 hours at room temperature,followed by centrifugation. The cell pellet was isolated phages wereeluted from the cells The supernatant was collected and added to E. coliTG-1 culture continued by the process described above. Two cell basedstrategies were employed to identify anti-HER3 antibodies:

-   -   a) Whole cell panning: In this strategy a variety of intact cell        lines were used as the antigens.    -   b) Differential whole cell panning: In this strategy the        antigens sequentially consisted of cells and recombinant HER3        proteins (see 1981.09 as an example). The cell based pannings        were performed as described above whilst solid phase panning        protocols were employed when recombinant proteins were utilized        as antigens. The washes were conducted using PBS (2-3×) and PBST        (2-3×).

(xi) RapMAT™ Library Generation and Pannings

In order to increase antibody binding affinity whilst maintaininglibrary diversity the second round output of both solution and solidphase pannings were entered into the RapMAT™ process whilst the thirdround output of the whole cell and differential whole cell panningstrategies were entered (Prassler et al., (2009) Immunotherapy; 1:571-583. RapMAT™ libraries were generated by sub-cloning Fab-encodinginserts of phages selected via panning into the display vectorpMORPH©25_bla_LHCand were further digested to either generate H-CDR2RapMAT™ libraries and L-CDR3 RapMAT™ libraries by using specificrestriction enzymes. The inserts were replaced with TRIM maturationcassettes (Vimekas et al., (1994) Nucleic Acids Research 22:5600-5607)for H-CDR2 or L-CDR3 according to pool composition. Library sizes wereestimated to range between 8×10⁶-1×10⁸ clones. RapMAT antibody-phagewere produced and subjected to two further rounds of solution, solidphase or cell based panning using the experimental methods describedpreviously.

Example 2: Transient Expression of Anti-HER3 IgG's

Suspension adapted HEK293-6E cells were cultivated in a BioWave20 to adensity of approximately 2×10⁶ viable cells/mL. The cells weretransiently transfected with the relevant sterile DNA: PEI-MIX andfurther cultivated. Seven days after transfection, cells were removed bycrossflow filtration using Fresenius filters (0.2 μm). The cell freematerial was concentrated with crossflow filtration using a 10 kDa cutoff filter (Fresenius) and the concentrate was sterile filtered througha stericup filter (0.22 μm). The sterile supernatant was stored at 4° C.

Example 3: Purification of Anti-HER3 IgG

The purification of IgG was performed on a AKTA 100 explorer Airchromatography system at 6° C. in a cooling cabinet, using a XK16/20column with 25 mL of self-packed MabSelect SuRe resin (all GEHealthcare). All flow rates were 3.5 mL/min, except for loading, at apressure limit of 5 bar. The column was equilibrated with 3 columnvolumes of PBS prior to loading the filtered fermentation supernatant at2.0 mL/min. The column was washed with 8 column volumes of PBS. IgG waseluted with a pH gradient, starting at 50 mM citrate/70 mM NaCl (pH4.5), going linearly down in 12 Column volumes to 50 mM citrate/70 mMNaCl (pH 2.5), followed by a 2 column volume constant step of the samepH 2.5 buffer. The IgG containing fractions were pooled and immediatelyneutralized and sterile filtered (Millipore Steriflip, 0.22 um). OD280was measured and the protein concentration calculated based on thesequence data. The pools were separately tested for aggregation(SEC-MALS) and purity (SDS-PAGE and MS).

Example 4: Expression and Purification of HuCAL®-Fab Antibodies in E.coli

Expression of Fab fragments encoded by pMORPH®X9_Fab_MH in TG-1 cellswas carried out in shaker flask cultures using 500 mL of 2×YT mediumsupplemented with 34 μg/mL chloramphenicol. Cultures were shaken at 30°C. until the OD600 nm reached 0.5. Expression was induced by addition of0.75 mM IPTG (isopropyl-β-D-thiogalactopyranoside) for 20 hours at 30°C. Cells were disrupted using lysozyme. His6-tagged (“His6” disclosed asSEQ ID NO: 495) Fab fragments were isolated via IMAC (Bio-Rad). Bufferexchange to 1× Dulbecco's PBS (pH 7.2) was performed using PD10 columns.Samples were sterile filtered (0.2 μm). Protein concentrations weredetermined by UV-spectrophotometry. The purity of the samples wasanalyzed in denaturing, reducing 15% SDS-PAGE. The homogeneity of Fabpreparations was determined in native state by size exclusionchromatography (HP-SEC) with calibration standards

Example 5: HER3 Antibody Affinity (K_(D)) Measurements by SolutionEquilibrium Titration (SET)

Affinity determination in solution was essentially performed aspreviously described (Friguet et al., (1985) J Immunol Methods77:305-19). In order to improve the sensitivity and accuracy of the SETmethod, it was transferred from classical ELISA to ECL based technology(Haenel et al., (2005) Anal biochem 339:182-84).

Unlabeled HER3-Tag (human, rat, mouse or cyno) described previously wasused for affinity determination by SET.

The data was evaluated with XLfit software (ID Business Solutions)applying customized fitting models. For K_(D) determination of each IgGthe following model was used (modified according to Piehler, et al(Piehler et al., (1997) J Immunol Methods 201:189-206).

$y = {\frac{2B_{\max}}{\lbrack{IgG}\rbrack}\left( {\frac{\lbrack{IgG}\rbrack}{2} - \frac{\left( {\frac{x + \lbrack{IgG}\rbrack + K_{D}}{2} - \sqrt{\frac{\left( {x + \lbrack{IgG}\rbrack + K_{D}} \right)^{2}}{4} - {x\lbrack{IgG}\rbrack}}} \right)^{2}}{2\lbrack{IgG}\rbrack}} \right)}$

[IgG]: applied total IgG concentration

x: applied total soluble antigen concentration (binding sites)

B_(max): maximal signal of IgG without antigen

K_(D): affinity

Example 6: Antibody Cell Binding Determination by FACS

The binding of antibodies to endogenous human antigen expressed on humancancer cells was accessed by FACS. In order to determine antibody EC₅₀values SK-Br-3 cells were harvested with accutase and diluted to 1×10⁶cells/mL in FACS buffer (PBS/3% FBS/0.2% NaN₃). 1×10⁵ cells/well wereadded to each well of a 96-well plate (Nunc) and centrifuged at 210 gfor 5 minutes at 4° C. before removing the supernatant. Serial dilutionsof test antibodies (diluted in 1:4 dilution steps with FACS buffer) wereadded to the pelleted cells and incubated for 1 hour on ice. The cellswere washed and pelleted three times with 100 μL FACS buffer. PEconjugated goat anti-human IgG (Jackson ImmunoResearch) diluted 1/200with FACS buffer were added to the cells and incubated on ice for 1hour. Additional washing steps were performed three times with 100 μLFACS buffer followed by centrifugation steps at 210 g for 5 minutes at4° C. Finally, cells were resuspended in 200 μL FACS buffer andfluorescence values were measured with a FACSArray (BD Biosciences). Theamount of cell surface bound anti-HER3 antibody was assessed bymeasuring the mean channel fluorescence.

Example 7: HER3 Domain and Mutant Binding

96-well Maxisorp plates (Nunc) were coated overnight at 4° C. with 200ng of the appropriate recombinant human protein (HER3-Tag, D1-2-Tag,D2-Tag, D3-4-Tag, D4-Tag, HER3 K267A-Tag, HER3 L268A-Tag, HER3K267A/L268A and a tagged irrelevant control). All wells were then washedthree times with PBS/0.1% Tween-20, blocked for one hour with PBS/1%BSA/0.1% Tween-20 and washed three times with PBS/0.1% Tween-20.Anti-HER3 antibodies were added to the relevant wells up to a finalconcentration of 10 μg/mL were added to the appropriate wells andincubated at room temperature for two hours. Plates were washed threetimes with PBS/0.1% Tween-20 prior to the addition of the appropriateperoxidase linked detection antibody diluted 1/10000 in PBS/1% BSA/0.1%Tween-20. The detection antibodies used were goat anti-mouse (Pierce,31432), rabbit anti-goat (Pierce, 31402) and goat anti-human (Pierce,31412). Plates were incubated at room temperature for one hour beforewashing three times with PBS/0.1% Tween-20. 100 μl TMB (3,3′, 5,5′tetramethyl benzidine) substrate solution (BioFx) was added to all wellsfor 6 minutes before stopping the reaction with 50 μl 2.5% H₂SO₄. Theextent of HER3 antibody binding to each recombinant protein wasdetermined by measuring the OD450 using a SpectraMax plate reader(Molecular Devices). Where appropriate, dose response curves wereanalyzed using Graphpad Prism.

Example 8: HER3 Epitope Mapping Using Hydrogen/Deuterium Exchange MassSpectrometry

Materials

D₂O buffer was made by dissolving 25 mM TBS (pH 7.5)/500 mM NaCl inheavy water (Sigma). The reduction solution was 50 mM formate buffer (pH4) 500 mM TCEP and the quenching solution 0.5% (v/v) trifluoroaceticacid (TFA) in water. Buffer A was 0.25% formic acid/10% methanol/10%ethylene glycol in water, and buffer B was 0.25% formic acid inacetonitrile. All chemicals were purchased from Sigma, and HPLC gradesolvents were from Fisher Scientific.

Liquid Handling and Chromatography

Automated hydrogen-deuterium exchange mass spectrometry (HDX MS)experiments were designed based upon methods and equipment described byWales et al., (2006) Anal. Chem. 78:1005-1014). In short, all liquidhandling operations used a Pal HTS liquid-handler (LEAP Technologies)housed in a refrigerated enclosure maintained at 2° C. A 6-portinjection valve and a wash station were mounted on the liquid-handlerrail and facilitated sample injection into the chromatographic systemand syringe washing. The chromatographic system, consisted of anadditional 10-port valve, a 2.1 mm×30 mm Poroszyme pepsin column(Applied Biosystems), a reverse-phase 0.5 mm×2 mm Cap Trap cartridge(Michrom Bioresources), and a self-packed electrospray emitter asanalytical column (100 μm×˜60 mm, Kinetex 2.6 μm C18, Phenomenex). The10-port valve head, the trap cartridge and the analytical column werehoused in a separate enclosure constructed from aluminum and maintainedat −5° C. by peltier stacks. Valves and columns were configured in sucha way as to allow in-line protein digestion, peptide desalting, andreversed-phase chromatography prior to introduction of the sample intothe electrospray ionization (ESI) source of the mass spectrometer(LTQ-Orbitrap, Thermo Scientific).

The fluid streams required for operation were provided by two separateHPLC pumps. The first HPLC (Surveyor MS pump, Thermo Scientific)delivered buffer A at a constant flow rate of 125 μL/min and was used totransfer sample through the immobilized pepsin cartridge onto thereversed-phase trap cartridge mounted across the 10-port valve. Afterthe loading and desalting period, the 10-port valve was switched toelute the sample with the help of a gradient pump (AQUITY UPLC, Waters)from the reversed-phase trap cartridge, through the analytical columnand into the ion source of the mass spectrometer. The immobilized enzymecartridge was isolated to waste during gradient elution. The gradientpump delivered linear gradient segments of 0 to 40% mobile phase B over35 minutes at 5 μL/min and 40 to 95% mobile phase B at 5 μL/min over 10minutes. The gradient flow from the pump was split at the 10-port valveusing a passive splitter so that the actual flow through the trapcartridge and analytical column for gradient elution was 1 μL/min. Theentire chromatographic run was 70 minutes long including washing andequilibration steps.

Mass Spectrometry

For the purpose of identification of proteolytic fragments resultingfrom online digestion several data-dependent MS/MS experiments wereperformed. For these acquisitions, tandem MS spectra were acquired withthe LTQ analyzer of the LTQ-Orbitrap hybrid mass spectrometer. Precursormass selection was based on MS scans acquired by the Orbitrap analyzer.Single stage MS acquisitions performed for the purpose of deuterationlevel determination were acquired at a resolution of 60,000 by theOrbitrap (over m/z 400-2000) analyzer.

Preparation of Protein and Protein:Fab Complexes

HER3 protein was prepared by diluting 50 μg HER3-Tag with 25 mM TBS (pH7.5)/500 mM NaCl to yield a final volume of 50 μL. Protein:Fab complexeswere prepared by mixing 50 μg HER3-Tag in a 1:1 molar ratio with theFab's studied. Protein:Fab mixtures were then diluted to a final volumeof 50 μL with 25 mM TBS (pH 7.5)/500 mM NaCl.

Protein:Fab complexes were prepared and allowed to incubate for at least2 hours at 4° C. Four 96-well plates containing sample, diluent, quench,and reduction solutions were loaded into the liquid-handler before thestart of each experimental. For on-exchange experiments 50 μL of HER3 orHER3:Fab complex was diluted with 150 μL D20 buffer. The mixture wasreduced by adding 200 μL reduction buffer for 1 minute before quenchingwith 600 μL of quench buffer. The total volume after all liquid handlingsteps was −1 mL. Once mixed, the quenched solution was injected into thechromatographic system where it was automatically digested, separatedand analyzed by LCMS. The average change in deuteration between sampleand control was calculated as the difference between the deuteriumuptake levels of the sample and control.

Data Processing

The Orbitrap RAW files were converted into mzXML files using an in-houseprogram (RawXtract). Subsequently, tandem MS acquisitions were searchedusing SEQUEST (Yates Lab, Scripps Research Institute, La Jolla, Calif.)and search results were automatically filtered using DTASelect 2.0(Yates Lab, Scripps Research Institute, La Jolla, Calif.). Using thepeptide sequence identifications, an in-house written program was usedto automatically extract single-ion chromatograms for each identifiedsequence and generate average spectra across the chromatographic peak.Average spectra were smoothed and centroided. The level of deuteriumuptake was taken as the difference in mass between a deuterated sampleand non-deuterated reference. Processed data was manually validated andadjusted to correct inaccuracies and errors from automated processingsteps. Deuterium uptake levels were assigned to each residue of theprotein sequence by delocalizing the deuterium content across eachpeptide (i.e., dividing the observed deuteration level by the number ofamino acids in that peptide). If a residue was covered by more than onepeptide, the normalized deuterium uptakes of all peptides covering thatresidue were averaged.

Example 9: X-Ray Crystallographic Structure Determination of the HumanHER3/MOR09823 Fab and Human HER3/MOR09825 Fab Complexes

The present example presents the crystal structure of full length HER3bound to the Fab fragment of MOR09823 and the Fab fragment of MOR09825,determined at 3.2 Å and 3.4 Å resolution, respectively. Tagged humanHER3 was further purified on a HiLoad 26/60 Superdex 200 PrepGradecolumn (GE Healthcare) equilibrated in PBS (pH 7.3). E. coli expressedMOR09823 and MOR09825 Fabs were isolated by lysing cells with lysozymeand His6-tagged (“His6” disclosed as SEQ ID NO: 495) Fab fragments werecaptured on a HisTrap_HP (GE Healthcare) column. MOR09823 Fab-fragmentswere further purified by gel filtration chromatography using a Superdex75 16/60 column (GE Healthcare) equilibrated in 25 mM Tris (pH 7.5), 150mM NaCl.

HER3 Fab complexes were prepared by mixing excess Fab with tagged HER3in a molar ratios of 1.3-1.8:1 (concentration estimated by absorbance at280 nm using calculated extinction coefficients of 0.9 and 1.4 (mg/ml)⁻¹cm⁻¹ for HER3 and Fab, respectively) and purifying the complexes on aSuperdex 200 10/300 column (GE Healthcare) equilibrated in 25 mM Tris(pH 7.5), 150 mM NaCl. Peak fractions were analyzed by SDS-PAGE andLCMS. For each complex, fractions containing both HER3 and Fab in anapproximate equimolar ratio were pooled and concentrated. HER3/MOR09823crystals were grown at 293K by sitting drop vapor diffusion from dropscontaining 150 nl HER3/MOR09823 complex and 150 nl of reservoir solution(100 mM sodium citrate pH 5.6, 20% PEG 4000 and 20% isopropanol).Crystals were transferred to reservoir solution containing additional 8%glycerol and flash cooled in liquid nitrogen. HER3/MOR09825 crystalswere grown at 293K by sitting drop vapor diffusion from drops containing150 nl HER3/MOR09825 complex and 150 nl of reservoir solution (100 mMbis-tris pH 6.5, 16% PEG 10,000). Crystals were transferred to 100 mMbis-tris pH 6.5, 18% PEG 10,000 and 22% glycerol and flash cooled inliquid nitrogen.

Data were collected at beamline 17-ID at the Advanced Photon Source(Argonne National Laboratory). HER3/MOR09823 Fab complex data wereprocessed and scaled at 3.2 Å using HKL2000 (HKL Research Inc) in spacegroup 1222 with cell dimensions a=124.16, b=139.44, c=180.25 Å, withgood statistics. The HER3/MOR09823 Fab structure was solved by molecularreplacement using Phaser (McCoy et al., (2007) J. Appl. Cryst.40:658-674) with fragments of a Fab and the published HER3 ECD structure1mb6 as search models. The final model, which contains 1 molecule of theHER3/MOR09823 Fab complex per asymmetric unit, was built in COOT (Emsley& Cowtan (2004) Acta Cryst. 60:2126-2132) and refined to R and R_(free)values of 19.0 and 24.5%, respectively, with an rmsd of 0.010 Å and 1.37for bond lengths and bond angles, respectively, using BUSTER (GlobalPhasing, LTD). Residues of HER3 that contain atoms within 5 Å of anyatom in MOR09823 Fab as identified in PyMOL (Schrödinger, LLC) arelisted in Tables 11 and 12. HER3/MOR09825 Fab complex data wereprocessed and scaled at 3.4 Å using autoPROC (Global Phasing, LTD) inspace group 1222 with cell dimensions a=124.23, b=140.94, c=180.25 Å,with good statistics. The HER3/MOR09825 Fab structure was solved bymolecular replacement using Phaser (McCoy et al., (2007) J. Appl. Cryst.40:658-674) with the HER3/MOR09823 Fab structure as a search model. Thefinal model, which contains 1 molecule of the HER3/MOR09825 Fab complexper asymmetric unit, was built in COOT (Emsley & Cowtan (2004) ActaCryst. 60:2126-2132) and refined to R and R_(free) values of 18.8 and24.9%, respectively, with an rmsd of 0.009 Å and 1.21° for bond lengthsand bond angles, respectively, using BUSTER (Global Phasing, LTD).Residues of HER3 that contain atoms within 5 Å of any atom in MOR09825Fab as identified in PyMOL (Schrödinger, LLC) are listed in Tables 13and 14.

Example 10: Phospho-HER3 In Vitro Cell Assays

MCF-7 cells were routinely maintained in DMEM/F12, 15 mM HEPES,L-glutamine, 10% FCS and SK-Br-3 in McCoy's 5a, 10% FCS, 1.5 mML-glutamine. Sub-confluent MCF7 or SK-Br-3 cells grown in complete mediawere harvested with accutase (PAA Laboratories) and resuspended in theappropriate growth media at a final concentration of 5×10¹ cells/mL. 100μL of cell suspension was then added to each well of a 96-well flatbottomed plate (Nunc) to give a final density of 5×10⁴ cells/well. MCF7cells were allowed to attach for approximately 3 hours before the mediawas exchanged for starvation media containing 0.5% FBS. All plates werethen incubated overnight at 37° C. prior to treatment with theappropriate concentration of HER3 antibodies (diluted in the appropriatemedia) for 80 minutes at 37° C. MCF7 cells were treated with 50 ng/mLneuregulin 1-β1 EGF domain (R&D Systems) for the final 20 minutes tostimulate HER3 phosphorylation. All media was gently aspirated and thecells washed with ice-cold PBS containing 1 mM CaCl₂) and 0.5 mM MgCl₂(Gibco). The cells were lysed by adding 50 μL ice-cold lysis buffer (20mM Tris (pH8.0)/137 mM NaCl/10% Glycerol/2 mM EDTA/1% NP-40/1 mM sodiumorthovanadate/, Aprotinin (10 μg/mL)/Leupeptin (10 μg/mL)) and incubatedon ice with shaking for 30 minutes. Lysates were then collected and spunat 1800 g for 15 minutes at 4° C. to remove cell debris. 20 μL of lysatewas added to a pre-prepared capture plate.

HER3 capture plates were generated using a carbon plate (MesoscaleDiscovery) coated overnight at 4° C. with 20 μL of 4 μg/mL MAB3481capture antibody (R&D Systems) diluted in PBS and subsequently blockedwith 3% bovine serum albumin in 1× Tris buffer (MesoscaleDiscovery)/0.1% Tween-20. HER3 was captured from the lysate byincubating the plate at room temperature for one hour with shakingbefore the lysate was aspirated and the wells washed with 1× Tris buffer(Mesoscale Discovery)/0.1% Tween-20. Phosphorylated HER3 was detectedusing 0.75 μg/mL biotinylated anti-phosphotyrosine antibody (R&DSystems) prepared in 1% BSA/1× Tris/0.1% Tween-20 by incubating withshaking at room temperature for 1 hour. The wells were washed four timeswith 1× Tris/0.1% Tween-20 and biotinylated proteins were detected byincubating with S-Tag labelled Streptavidin (Mesoscale Discovery)diluted in 1% BSA/1× Tris/0.1% Tween-20 for one hour at roomtemperature. Each well was aspirated and washed four times with 1×Tris/0.1% Tween-20 before adding 20 μL of Read buffer T with surfactant(Mesoscale Discovery) and the signal quantified using a Mesoscale SectorImager. Antibodies MOR06391 or MOR03207 were included in signallingexperiments as isotype controls.

Example 11: Phospho-Akt (S473) In Vitro Cell Assays

Sub-confluent SK-Br-3 and BT-474 cells grown in complete media wereharvested with accutase (PAA Laboratories) and resuspended in theappropriate growth media at a final concentration of 5×10⁵ cells/mL. 100μL of cell suspension was then added to each well of a 96-well flatbottomed plate (Nunc) to yield a final density of 5×10⁴ cells/well. Allplates were then incubated overnight at 37° C. prior to treatment withthe appropriate concentration of HER3 antibodies (diluted in theappropriate media) for 80 minutes at 37° C. All media was gentlyaspirated and the cells washed with ice-cold PBS containing 1 mM CaCl₂)and 0.5 mM MgCl₂ (Gibco). The cells were lysed by adding 50 μL ice-coldlysis buffer (20 mM Tris (pH8.0)/137 mM NaCl/10% Glycerol/2 mM EDTA/1%NP-40/1 mM sodium orthovanadate/Aprotinin (10 μg/mL)/Leupeptin (10μg/mL)) and incubated on ice with shaking for 30 minutes. Lysates werethen collected and spun at 1800 g for 15 minutes at 4° C. to remove celldebris. 20 μL of lysate was added to a multi-spot 384-well Phospho-Aktcarbon plate (Mesoscale Discovery) that had previously been blocked with3% BSA/1× Tris/0.1% Tween-20. The plate was incubated at roomtemperature for two hours with shaking before the lysate was aspiratedand the wells washed four times with 1× Tris buffer (MesoscaleDiscovery)/0.1% Tween-20. Phosphorylated Akt was detected using 20 μL ofSULFO-TAG anti-phospho-Akt (S473) antibody (Mesoscale Discovery) diluted50-fold in 1% BSA/1× Tris/0.1% Tween-20 by incubating with shaking atroom temperature for 2 hours. The wells were washed four times with 1×Tris/0.1% Tween-20 before adding 20 μL of Read buffer T with surfactant(Mesoscale Discovery) and the signal quantified using a Mesoscale SectorImager. Antibodies MOR06391 or MOR03207 were included in signallingexperiments as isotype controls.

Example 12: Cell-Line Proliferation Assays

SK-Br-3 cells were routinely cultured in McCoy's 5 Å medium modified,supplemented with 10% fetal bovine serum and BT-474 cells were culturedin DMEM supplemented with 10% FBS. Sub-confluent cells were trypsinized,washed with PBS, diluted to 5×10⁴ cells/mL with growth media and platedin 96-well clear bottom black plates (Costar 3904) at a density of 5000cells/well. The cells were incubated overnight at 37° C. before addingthe appropriate concentration of HER3 antibody (typical finalconcentrations of 10 or 1 μg/mL). The plates were returned to theincubator for 6 days before assessing cell viability using CellTiter-Glo(Promega). 100 μL of CellTiter-Glo solution was added to each well andincubated at room temperature with gentle shaking for 10 minutes. Theamount of luminescence was determined using a SpectraMax plate reader(Molecular Devices). The extent of growth inhibition obtained with eachantibody was calculated by comparing the luminescence values obtainedwith each HER3 antibody to a standard isotype control antibody(MOR06391).

For proliferation assays MCF-7 cells were routinely cultured in DMEM/F12(1:1) containing 4 mM L-Glutamine/15 mM HEPES/10% FBS. Sub-confluentcells were trypsinized, washed with PBS and diluted to 1×10⁵ cells/mLwith DMEM/F12 (1:1) containing 4 mM L-Glutamine/15 mM HEPES/10 μg/mLHuman Transferrin/0.2% BSA. Cells were plated in 96-well clear bottomblack plates (Costar) at a density of 5000 cells/well. The appropriateconcentration of HER3 antibody (typical final concentrations of 10 or 1μg/mL) was then added. 10 ng/mL of NRG1-β1 EGF domain (R&D Systems) wasalso added to the appropriate wells to stimulate cell growth. The plateswere returned to the incubator for 6 days before assessing cellviability using CellTiter-Glo (Promega). The extent of growth inhibitionobtained with each antibody was calculated by subtracting the background(no neuregulin) luminescence values and comparing the resulting valuesobtained with each anti-HER3 antibody to a standard isotype controlantibody (MOR06391).

Example 13: Ligand Blocking Cell Assays

MCF-7 cells cultured in MEM supplemented with 10% FBS and 1 μg/mLinsulin (Sigma) were rinsed and collected in a small volume of FACSmaxcell dissociation buffer (Genlantis) prior to the addition of 5 mL ofFACS buffer (PBS/1% FBS/0.1% sodium azide). The cell density was countedand adjusted to a final concentration of 1×10⁶ cells/mL. 100 μl of cellsuspension was added to each well of a 96-well plate and the cellspelleted via centrifugation (220 g, 3 minutes, 4° C.). Cell pellets wereresuspended in 100 μL of the appropriate test antibodies diluted in FACSbuffer (typical final antibody concentrations ranged from 100 to 0.1 nM)and the plate incubated on ice for 45 minutes. The ligand blockingantibody MAB3481 (R&D Systems) was included as a positive control. Cellswere washed twice with staining buffer prior to adding 10 nM NRG1-01 EGFdomain (R&D Systems) diluted in FACS buffer and incubating on ice for 45minutes. Cells were washed twice with staining buffer and boundneuregulin detected by incubating the cells with 10 nM anti-humanNRG1-R1 EGF domain antibody (R&D Systems) on ice for 45 minutes. Cellswere washed twice with staining buffer and incubated on ice for 45minutes with PE-linked anti-goat antibody (Jackson ImmunoResearch)diluted 1/500 with FACS buffer. Cells were then pelleted viacentrifugation and the pellet resuspended in 200 μL FACS buffer. Toquantify each sample 10,000 live cells were counted on a LSR II FlowCytometer (BD Biosciences) and the amount of cell surface boundneuregulin was assessed by measuring the mean channel fluorescence.

Example 14: Ligand Blocking Biochemical Assay

The present method includes utility of a Surface plasmon resonance(SPR)-based biosensor (Biacore, GE Healthcare, Uppsala, Sweden) toexamine the ability of HER3/antibody complexes to bind neuregulin.

Biacore™ m utilizes the phenomenon of surface plasmon resonance (SPR) todetect and measure binding interactions. In a typical Biacoreexperiment, one of the interacting molecules (neuregulin) is immobilizedon a matrix while the interacting partner (HER3) is flowed over thesurface. A binding interaction results in an increase in mass on thesensor surface and a corresponding direct change in the refractive indexof the medium in the vicinity of the sensor surface. Changes inrefractive index or signal are recorded in resonance units (R.U.) Signalchanges due to association and dissociation of complexes are monitoredin a non-invasive manner, continuously and in real-time, the results ofwhich are reported in the form of a sensorgram.

Biacore™ T100 (GE Healthcare, Uppsala, Sweden) was used to conduct allexperiments reported herein. Sensor surface preparation and interactionanalyses were performed at 25° C. Buffer and Biacore reagents werepurchased from GE Healthcare. Running buffer containing 10 mM Hepes,pH7.4/150 mM NaCl, 0.05% P20, 0.5% BSA was utilized throughout theassay.

NRG-1β1 extracellular domain (R&D Systems) was incubated on ice for 45minutes with EZ-link Sulfo-NHS-LC-LC-Biotin (Pierce) at a molar ratio of5:1. The reaction was quenched via the addition excess ethanolamine anduncoupled biotin removed from the biotinylated-NRG using desalt spincolumns (Zeba). Biotinylated-NRG was captured onto a sensor chip CAPpre-immobilized with approximately 3000 R.U. of ssDNA-streptavidin(Biotin CAPture kit) to yield neuregulin surface densities in the range400-600 R.U. A reference flowcell was generated by omittingbiotinylated-NRG from the injection steps such that onlyssDNA-streptavidin was present on the flowcell surface.

HER3/antibody complexes were generated by incubating 10 nM human HER3-Fcwith increasing concentrations (0-50 nM) of the appropriate testantibody for 15 minutes at room temperature prior to incubating in theBiacore™ at 10° C. Interaction analyses were performed by injectingHER3/antibody complexes over reference and neuregulin surfaces in seriesfor 180 seconds at a flow-rate of 60 L/min. Complex dissociation wasmonitored for 180 seconds at a flow rate of 60 L/min. Surfaceregeneration was performed at the end of each analysis cycle using a 120second injection of 8M guanidine: 1M NaOH (3:1) followed by a 120 secondinjection of 30% acetonitrile/0.25M NaOH at a flow rate of 30 L/min.

Example 15: In Vivo PD Studies

BxPC3 and BT-474 cells were cultured and implanted in female athymicnu/nu Balb/C mice (Harlan Laboratories) as described in Examples 16 and17.

Once tumors had reached an appropriate size, animals were examined fortumor quality. Animals with ulcerated tumors or animal with fluid-filledtumors were excluded from the study. The remaining animals were dosedintravenously with antibody via lateral tail vein injection. At thegiven time points, animals were euthanized via CO₂ asphyxiation andwhole blood was collected via cardiac puncture and placed into a 1.5 mLEppendorf collection tube. Tumor tissue was immediately dissected,placed into a screw-top polypropylene sample tube and snap frozen inliquid nitrogen. Tissue was stored at −80° C. until lysates wereprepared.

Example 16: In Vivo BT-474 Efficacy Studies

BT-474 cells were cultured in DMEM containing 10% heat-inactivated fetalbovine serum without antibiotics until the time of implantation.

One day before cell inoculation, female athymic nu/nu Balb/C mice(Harlan Laboratories) were implanted subcutaneously with a sustainedrelease 17β-estradiol pellet (Innovative Research of America) tomaintain serum estrogen levels. One day after 17β-estradiol pelletimplantation, 5×10⁶ cells were injected orthotopically into the 4^(th)mammary fatpad in a suspension containing 50% phenol red-free matrigel(BD Biosciences) in Hank's balanced salt solution. The total injectionvolume containing cells in suspension was 200 μL. 20 days following cellimplantation animals with a tumor volume of approximately 200 mm³ wereenrolled in the efficacy study. In general, a total of 10 animals pergroup were enrolled in efficacy studies.

For single-agent studies, animals were dosed intravenously via lateraltail vein injection with either MOR10701 or MOR10703. An initial loadingdose of 40 mg/kg was given for the first dose. After the initial dose,animals were on a 20 mg/kg, every other day schedule for the duration ofthe study. For combination studies, animals were dosed with eitherMOR10701 or MOR10703 (20 mg/kg, iv, q2d) and a sub-optimal dose oftrastuzumab (1 mg/kg, iv, 2qw). For the duration of the studies, tumorvolume was measured by calipering twice per week. Percenttreatment/control (T/C) values were calculated using the followingformula:

% T/C=100×ΔT/ΔC if ΔT>0

where:

T=mean tumor volume of the drug-treated group on the final day of thestudy;

ΔT=mean tumor volume of the drug-treated group on the final day of thestudy−mean tumor volume of the drug-treated group on initial day ofdosing;

C=mean tumor volume of the control group on the final day of the study;and

ΔC=mean tumor volume of the control group on the final day of thestudy−mean tumor volume of the control group on initial day of dosing.

Body weight was measured twice per week and dose was body weightadjusted. The % change in body weight was calculated as(BW_(current)−BW_(initial))/(BW_(initial))×100. Data is presented aspercent body weight change from the day of treatment initiation.

All data were expressed as mean standard error of the mean (SEM). Deltatumor volume and body weight were used for statistical analysis. Betweengroups comparisons were carried out using a one-way ANOVA followed by apost hoc Tukey. For all statistical evaluations the level ofsignificance was set at p<0.05. Significance compared to the vehiclecontrol group is reported.

Example 17: In Vivo BxPC3 Efficacy Studies

BxPC3 cells were cultured in RPMI-1640 medium containing 10%heat-inactivated fetal bovine serum without antibiotics until the timeof implantation.

Female athymic nu/nu Balb/C mice (Harlan Laboratories) were implantedsubcutaneously with 10×10⁶ cells in a mixture of 50% phosphate bufferedsaline with 50% matrigel. The total injection volume containing cells insuspension was 200 μL. Once tumors had reached approximately 200 mm³ insize, animals were enrolled in the efficacy study. In general, a totalof 10 animals per group were enrolled in studies. Animals were excludedfrom enrollment if they exhibited unusual tumor growth characteristicsprior to enrollment.

Animals were dosed intravenously via lateral tail vein injection. Aninitial loading dose of 40 mg/kg was given for the first dose. After theinitial dose, animals were on a 20 mg/kg, every other day schedule forthe duration of the study (25 days under treatment). Tumor volume andT/C values were calculated as previously detailed.

Example 18: Phospho-Akt (S473) In Vivo PD Assays

Approximately 50 mm³ frozen tumor (e.g. BT-474 or BXPC-3) tissue wasthawed on ice and 100-300 μL of T-PER buffer (Pierce) containingphosphatase (Roche) and protease inhibitors (Roche) was added to eachsample. The volume of lysis buffer added was dependent upon the size ofthe tumor sample. The tissue was broken down using a 1.5 mL pestle(Fisher Scientific) and the resultant suspensions were incubated on icefor 15 minutes before being frozen overnight at −80° C. Samples werethawed and spun for 15 minutes at 13000 g, 4° C. prior to quantifyingthe supernatant protein concentration by BCA assay (Thermo Scientific).Tissue supernatants were diluted with lysis buffer (Mesoscale Discovery)and 25 μg added to a multi-spot 96-well Phospho-Akt carbon plate(Mesoscale Discovery) that had previously been blocked with BlockingSolution-A (Mesoscale Discovery). The plate was incubated at roomtemperature for one hour with shaking before the lysate was aspiratedand the wells washed four times with Tris Wash buffer (MesoscaleDiscovery). Phosphorylated Akt was detected using 25 μL of SULFO-TAGanti-phospho-Akt (S473) antibody (Mesoscale Discovery) diluted inantibody dilution buffer by incubating with shaking at room temperaturefor one hour. The wells were washed four times with Tris Wash bufferbefore adding 150 μL of Read buffer T (with surfactant) (MesoscaleDiscovery) and the signal quantified using a Mesoscale Sector Imager.

Example 19: Phospho HER3 (Y1197) In Vivo PD Assays

Approximately 50 mm³ frozen tumor (e.g. BXPC-3) tissue was thawed on iceand 100-300 μL of T-PER buffer (Pierce) containing phosphatase (Roche)and protease inhibitors (Roche) was added to each sample. The tissue wasbroken down using a 1.5 mL pestle (Fisher Scientific) and the resultantsuspensions were incubated on ice for 15 minutes before being frozenovernight at −80° C. Samples were thawed and spun for 15 minutes at13000 g, 4° C. prior to quantifying the supernatant proteinconcentration by BCA assay (Thermo Scientific). Tissue supernatants werediluted with lysis buffer and 150 μg added to a multi-spot 96-wellcarbon plate (Mesoscale Discovery) that had previously been coatedovernight with 4 μg/mL MAB3481 (R&D Systems) and blocked with 3% milk.The plate was incubated at room temperature for two hours with shakingbefore the lysate was aspirated and the wells washed four times withTris Wash buffer (Mesoscale Discovery). Phosphorylated HER3 was boundusing anti-HER3 pY1197 diluted 1/8000 with blocking buffer. Followingincubation at room temperature for one hour the wells were washed withTris Wash buffer and the anti-pY1197 antibody detected using S-Taglabelled anti-rabbit antibody (Mesoscale Discovery) diluted 1/1000 inblocking buffer by incubating with shaking at room temperature for onehour. The wells were washed four times with Tris Wash buffer beforeadding 150 μl of 1/4 diluted Read buffer T (with surfactant) (MesoscaleDiscovery) and the signal quantified using a Mesoscale Sector Imager.

Example 20: In Vitro Drug Combination Studies

To assess the ability of HER3-targeted antibodies to combine withtargeted therapies MOR09825 or MORI0703 were combined with trastuzumab,lapatinib, BEZ235, BKM120, BYL719, RAD001, erlotinib and cetuximab incell viability assays. Approximately 1000-1500 SK-Br-3 (McCoy's),MDA-MB-453 (RPMI), FaDu (EMEM) or L3.3 (RPMI) cells were seeded into384-well plates in the appropriate culture media supplemented with 2%FBS and allowed to adhere overnight at 37° C. The appropriate drugcombinations (typical final drug concentrations for lapatinib, BKM120,and BYL719 ranged from 3 μM to 13 nM; for RAD001 ranged from 27 nM to0.0041 nM; for erlotinib ranged from 1 μM to 0.0025 nM; for MOR1073ranged from 100 nm to 0.01 nm; for cetuximab ranged from 100 nM to0.0015 nM; and for trastuzumab ranged from 300 nM to 0.046 nM)) weresubsequently added to the wells such that each plate contained a doseresponse curve of each drug in a two-dimensional matrix. The plates werereturned to the incubator for 3-6 days before assessing cell viabilityusing CellTiter-Glo (Promega). CellTiter-Glo solution was added to eachwell and incubated at room temperature with gentle shaking for 10minutes. The amount of luminescence was determined using a SpectraMaxplate reader (Molecular Devices). The extent of growth inhibitionobtained with each combination was calculated and combination activityhighlighted using the Loewe additivity model.

Example 21: In Vivo Drug Combination Studies in L3.3 Cells

Pancreatic L3.3 cells were cultured in DMEM medium containing 10%heat-inactivated fetal bovine serum until the time of implantation.Female Foxn1 nude mice (Harlan Laboratories) were implantedsubcutaneously with 3×10⁶ cells in FBS free DMEM. The total injectionvolume containing cells in suspension was 100 μL. 12 days following cellimplantation, animals were enrolled in the efficacy study with a meantumor volume of approximately 100 mm³ for all groups. In general, atotal of 8 animals per group were enrolled in studies. Animals wereexcluded from enrollment if they exhibited unusual tumor growthcharacteristics prior to enrollment.

Animals were dosed intravenously with MOR10703 via lateral tail veininjection on a 20 mg/kg, every other day schedule for the duration ofthe study (14 days under treatment). Erlotinib was dosed at 50 mg/kg(PO) on a daily schedule either as a single-agent or in combination withR10703. Tumor volume and T/C values were calculated as previouslydetailed.

Results and Discussion

Collectively, these results show that a class of antibodies bind toamino acid residues within domain 2 and domain 4 of a conformationalepitope of HER3 and stabilizes HER3 in an inactive or closedconformation. Binding of these antibodies inhibits both ligand-dependentand ligand-independent signaling. These antibodies are also able to bindconcurrently with a HER3 ligand.

(i) Affinity Determination

Antibody affinity was determined by solution equilibrium titration (SET)as described above. The results are summarized in Table 9 and exampletitration curves for MOR10701 are contained in FIG. 1. The data indicatethat a number of antibodies were identified that tightly bound human,cyno, rat and murine R3.

TABLE 9 K_(D) values of anti-HER3 IgGs as determined by solutionequilibrium titration (SET). Hu (human), Cy (cynomolgus), Mu (murine)and ra (rat) SET K_(D) (pM) hu cy mu ra MOR# HER3-Tag HER3-Tag HER3-TagHER3-Tag 09823 9 4 2 11 09824 3 3 2 7 09825 25 56 24 96 09974 350 200120 n.d. 10701 4 4 6 10 10702 3 3 5 6 10703 26 23 20 40 12609 10 n.d n.dn.d 12610 37 n.d n.d n.d 10703 N52S 57 n.d n.d n.d 10703 N52G 60 n.d n.dn.d 10703_A50V_N52S 16 n.d n.d n.d 10703_A50V_N52G 22 n.d n.d n.d 10701R55G 18 n.d n.d n.d 10701 R55K 11 n.d n.d n.d

(ii) SK-Br-3 Cell EC₅₀ Determination

The ability of the identified antibodies to bind HER3 expressing cellswas determined by calculating EC₅₀ values for their binding to the HER2amplified cell line SK-Br-3 (see FIG. 2 and Table 10).

TABLE 10 FACS EC₅₀ values of anti-HER3 IgG on SK-Br-3 cells. MOR#SK-Br-3 FACS EC₅₀ (pM) 09823  630 09824  324 09825  839 09974 n.d. 10701n.d. 10702 n.d. 10703 2454 n.d. (not determined)

(iii) HER3 Domain Binding

A subset of anti-HER3 antibodies were characterized for their ability tobind the various extracellular domains of human HER3 in an ELISA assay.To achieve this, the extracellular domain of HER3 was divided into itsfour constitutive domains and various combinations of these domains werecloned, expressed and purified as independent proteins as describedabove. Using this strategy the following domains were successfullygenerated as soluble proteins: domains 1 and 2 (D1-2), domain 2 (D2),domains 3 and 4 (D3-4) and domain 4 (D4). A number of internallygenerated mouse anti-human HER3 antibodies (8D7, 1F5 and 8P2) were alsotested as positive controls to demonstrate the integrity of eachisolated domain.

As shown in FIG. 3 MOR09823 and MOR09825 were both observed tosuccessfully bind the HER3 extracellular domain, but little binding tothe isolated domains was observed in this assay with these antibodies.There are several possible explanations for this binding pattern:

a) MOR09823 and MOR09825 may bind a linear epitope that spans a domainboundary thus part of the binding epitope would be lost when the domainswere expressed as isolated proteins.

b) MOR09823 and MOR09825 may bind a non-linear epitope that bridgesmultiple domains. Consequently, separation of HER3 into its componentunits may destroy the binding site.

c) The shape/conformation of HER3 may be a component of the binding ofMOR09823 and MOR09825 to HER3 such that only the full-lengthextracellular domain of HER3 is capable of adopting thisshape/conformation whilst the isolated domains cannot fully assume thisconformation.

(vi) HER3 Epitope Mapping Using Hydrogen/Deuterium Exchange MassSpectrometry

The HER3 epitope was further explored by HDX-MS analysis of HER3 ECD inthe presence and absence of Fab versions of MOR09823, MOR09824, MOR09825and MOR09974. FIG. 4A shows that in the absence of bound Fab,approximately 69% of the HER3 ECD sequence was covered by at least onepeptide. Gaps in coverage may be due to glycosylation of residues withinthese regions or insufficient reduction of disulphide bonds in cysteinerich regions, which is particularly apparent in domain 2. Interestingly,although each Fab yielded individual protection patterns, one region ofstrong protection was consistently observed with MOR09823, MOR09824,MOR09825 and MOR09974 (see FIG. 4B) indicating that these highly relatedfamily of antibodies bind HER3 in an identical manner. The strongestprotection was observed for domain 2 residues 269-286(TFQLEPNPHTKYQYGGVC) (SEQ ID NO: 496) indicating that residues in thisvicinity may be important for mAb binding. Mapping of the Fab protectedresidues onto the published HER3 crystal structure (Cho & Leahy, (2002)Science 297:1330-1333) highlights that residues 269-286 are within andproximal to a functionally important β-hairpin loop within domain 2 (seeFIG. 4C).

(vii) HER3/MOR09823 Crystal Structure

The 3.2 Å resolution x-ray crystal structure of MOR09823 Fab fragmentbound to the HER3 extracellular domain was solved to further define theHER3 epitope that is recognized by this family of related antibodies(see FIG. 5A). In addition, the 3.4 Å structure of MOR09825 Fab fragmentbound to human HER3 was resolved. In both the MOR09823/HER3 andMOR09825/HER3 crystal structures, HER3 is in the tethered (inactive)conformation (see FIGS. 5A, B, C and D). This conformation ischaracterized by a significant interaction interface between domains 2and 4 mediated by a β-hairpin dimerization loop in domain 2. Theobserved conformation of HER3 is similar to that previously described byCho et al. (Cho & Leahy, (2002), Science 297:1330-1333) who publishedthe crystal structure of the HER3 extra-cellular domain in the absenceof neuregulin. Since neuregulin can activate HER3, the tetheredconformation of HER3 is presumed to be inactive. Similar tetheredconformations have also been observed when the related EGFR familymembers HER4 (Bouyain et al., (2005) Proc. Natl. Acad. Sci. USA,102:15024-15029) and HER1 (Ferguson et al., (2003) Molec. Cell11:507-517) have been crystallized.

The spatial relationships between domains 1 to 4 of HER3 in the inactive(tethered) state are significantly different from that of the extended(active) state. This finding is based upon the crystal structures of therelated EGFR family members HER2 and ligand-bound HER1 (Cho et al.,(2003) Nature 421:756-760; Ogiso et al., (2002) Cell 110:775-787;Garrett et al., (2002) Cell 110:763-773) both of which are in anextended (active) state. In the extended state, the domain 2 β-hairpindimerization loop is released from its inhibitory interaction with 4 andis thus free to interact with its dimerization partner proteins. Thus,the domain 2 β-hairpin dimerization loop is functionally important bothin maintaining the tethered (inactive) state and in mediatingdimerization of EGF receptors in the extended state, leading toactivation of the intracellular kinase domain. The MOR09823/HER3 andMOR09825/HER3 crystal structures (see FIG. 5) therefore suggest thatboth MOR09823 and MOR09825 function by stabilizing the inactiveconformation of HER3.

The crystal structure also revealed that the HER3 epitope recognized byboth MOR09823 and MOR09825 is a non-linear epitope that includesresidues from both domains 2 and 4 (see FIGS. 5C and D, Tables 11, 12,13 and 14). The HER3 epitope recognized by this family of highly relatedantibodies can therefore be defined as:

Domain 2: residues 265-277, 315

Domain 4 residues: 571, 582-584, 596-597, 600-602, 609-615

Binding of both domains 2 and 4 by MOR09823 or MOR09825 wouldconsequently stabilize the tethered conformation of HER3 thusantagonizing its ability to signal.

The MOR09823/MOR09825 binding mode observed in the crystal structure isconsistent with our other epitope mapping studies. Specifically, theELISA domain binding experiments demonstrate that the affinity ofMOR09823 and MOR09825 are significantly greater for the intact HER3extracellular protein than for any isolated domains (e.g. D1, D1-D2, D3,or D3-D4 fragments) (see FIG. 3). There is also agreement with the HER3HDX-MS data (see FIG. 4B), which identifies domain 2 β-hairpin as partof the antibody recognition epitope. Finally, both crystal structuresindicate that the ligand-binding surface of HER3, which has been mappedby analogy to HER1 to domains 1 and 3 (Ogiso et al., (2002) Cell,110:775-787; Garrett et al., (2002) Cell, 110:763-773) is not occludedby either MOR09823 or MOR09825 binding (see FIG. 5B). This is consistentwith our findings that neither MOR09823 nor MOR09825 block neuregulinbinding to MCF7 cells (see FIG. 9) and that HER3/MOR09823 complexes canbind to immobilized neuregulin in biacore studies (see FIG. 10).

TABLE 11 Interactions between MOR09823 Fab heavy chain and human HER3.Fab VH residues are numbered based upon their linear amino acid sequence(SEQ ID NO: 15). HER3 residues are numbered based upon NP_001973. HER3residues shown have at least one atom within 5Å of an atom in theMOR09823 Fab. MOR09823 Fab Human HER3 Residue Number Chain ResidueNumber Domain Ser  30 VH Pro 276 2 Ser  31 VH Pro 274 2 Asn 275 2 Pro276 2 Tyr  32 VH Pro 276 2 His 277 2 Ala  33 VH Asn 266 2 Leu 268 2 Ser 35 VH Leu 268 2 Val  50 VH Leu 268 2 Thr 269 2 Gly  52 VH Glu 273 2 Thr269 2 Ala  53 VH Glu 273 2 Pro 274 2 Val  54 VH Glu 273 2 Tyr  58 VH Pro583 4 Asp 571 4 His 584 4 Thr 269 2 Gln 271 2 Asn  73 VH Asn 315 2 Ser 74 VH Asn 315 2 Trp  98 VH Leu 268 2 Lys 267 2 Asn 266 2 Asp 100 VH Ala596 4 Lys 597 4 Pro 276 2 His 277 2 Glu 101 VH Lys 267 2 Lys 597 4 Phe103 VH Leu 268 2

TABLE 12 Interactions between MOR09823 Fab light chain and human HER3.Fab VL residues are numbered based upon their linear amino acid sequence(SEQ ID NO: 14). HER3 residues are numbered based upon NP_001973. HER3residues shown have at least one atom within 5Å of an atom in theMOR09823 Fab. MOR09823 Fab Human HER3 Residue Number Chain ResidueNumber Domain Gln 27 VL Arg 611 4 Glu 609 4 Gly 28 VL Arg 611 4 Pro 6124 Ile 29 VL Pro 612 4 Ser 30 VL Pro 612 4 Cys 613 4 His 614 4 Glu 615 4Asn 31 VL Glu 615 4 Cys 613 4 Trp 32 VL Lys 267 2 Tyr 265 2 Pro 612 4Cys 613 4 Ile 600 4 Lys 602 4 Tyr 49 VL Lys 597 4 Gly 66 VL Glu 615 4Ser 67 VL His 614 4 Glu 615 4 Gln 89 VL Leu 268 2 Tyr 91 VL Lys 267 2Leu 268 2 Phe 270 2 Ser 92 VL Phe 270 2 Lys 602 4 Pro 612 4 Ser 93 VLPhe 270 2 Glu 609 4 Phe 94 VL Phe 270 2 Leu 268 2 Gly 582 4 Pro 583 4Thr 96 VL Leu 268 2

TABLE 13 Interactions between MOR09825 Fab heavy chain and human HER3.Fab VH residues are numbered based upon their linear amino acid sequence(SEQ ID NO: 51). HER3 residues are numbered based upon NP_001973. HER3residues shown have at least one atom within 5Å of an atom in theMOR09825 Fab. MOR09825 Fab Human HER3 Residue Number Chain ResidueNumber Domain Ser 30 VH Asn 315 2 Ser 31 VH Pro 274 2 Pro 276 2 Tyr 32VH Pro 276 2 His 277 2 Ala 33 VH Asn 266 2 Thr 269 2 Ser 35 VH Leu 268 2Trp 47 VH Leu 268 2 Ala 50 VH Leu 268 2 Asn 52 VH Glu 273 2 Gln 271 2Thr 269 2 Ser 53 VH Glu 273 2 Pro 274 2 Gln 54 VH Glu 273 2 Pro 274 2Ser 57 VH Gln 271 2 Tyr 59 VH Pro 583 4 Asp 571 4 His 584 4 Thr 269 2Gln 271 2 Asn 74 VH Asn 315 2 Trp 99 VH Leu 268 2 Lys 267 2 Asn 266 2Asp 101  VH Ala 596 4 Lys 597 4 Pro 276 2 His 277 2 Glu 102  VH Lys 2672 Lys 597 4 Phe 104  VH Leu 268 2

TABLE 14 Interactions between MOR09825 Fab light chain and human HER3.Fab VL residues are numbered based upon their linear amino acid sequence(SEQ ID NO: 50). HER3 residues are numbered based upon NP_001973. HER3residues shown have at least one atom within 5Å of an atom in theMOR09825 Fab. MOR09825 Fab Human HER3 Residue Number Chain ResidueNumber Domain Gln 27 VL Arg 611 4 Gly 28 VL Arg 611 4 Pro 612 4 Ile 29VL Pro 612 4 Ser 30 VL Pro 612 4 Cys 613 4 His 614 4 Glu 615 4 Asn 31 VLGlu 615 4 His 614 4 Cys 613 4 Trp 32 VL Lys 267 2 Tyr 265 2 Pro 612 4Cys 613 4 Ile 600 4 Lys 602 4 Tyr 49 VL Lys 597 4 Gly 66 VL Glu 615 4Ser 67 VL His 614 4 Glu 615 4 Gln 89 VL Leu 268 2 Tyr 91 VL Lys 267 2Leu 268 2 Phe 270 2 Ser 92 VL Phe 270 2 Lys 602 4 Pro 612 4 Arg 611 4Ser 93 VL Phe 270 2 Glu 609 4 Phe 94 VL Phe 270 2 Gly 582 4 Pro 583 4Thr 96 VL Leu 268 2

Visual inspection of the MOR09823/MOR09825 crystal structureshighlighted that HER3 residues Lys267 and Leu268 formed multipleinteractions with various antibody CDR's suggesting that they may beimportant for antibody binding. Consequently, Lys267 and/or Leu268 weremutated to alanine, expressed and the resultant recombinant proteinspurified in order to assess their impact upon antibody binding. ELISAbinding assays indicated that mutation of either Lys267 or Leu268abolished MORI0703 binding to HER3 (FIG. 5F) suggesting that bothresidues are an integral part of the HER3 epitope and thus supportingthe proposed interactions between MOR09823/MOR09825 and HER3.

(viii) Inhibition of Cell Signaling

To ascertain the effect of anti-HER3 antibodies upon ligand dependentHER3 activity MCF7 cells were incubated with IgG prior to stimulationwith neuregulin. Example inhibition curves are illustrated in FIG. 6Aand summarized in Table 15. The effect of anti-HER3 antibodies uponHER2-mediated HER3 activation was also studied using the HER2 amplifiedcell line SK-Br-3 (FIG. 6B and Table 15).

TABLE 15 pHER3 IC₅₀ and extent of inhibition values of anti-HER3 IgG inMCF7, and SK-Br-3 cells. MCF7 pHER3 SK-Br-3 pHER3 IC₅₀ % IC₅₀ % MOR#(pM) inhibition (pM) inhibition 09823 181 89 56 59 09824 103 91 110 6409825 399 80 169 66 09974 3066 69 1928 67 10701 n.d. n.d. 370 74 10702n.d. n.d. n.d. n.d. 10703 333 80 167 69 12609 5 86 241 71 12610 126 84192 75

To determine whether inhibition of HER3 activity impacted downstreamcell signaling Akt, phosphorylation was also measured in HER2 amplifiedcells following treatment with anti-HER3 antibodies (see FIG. 7 andTable 16).

TABLE 16 pAkt (S⁴⁷³) IC₅₀ and extent of inhibition values of anti-HER3IgG in SK-Br-3 BT-474 and MCF7 cells. SK-Br-3 pAkt BT-474 pAkt MCF7 pAktIC₅₀ % % IC₅₀ % MOR# (pM) inhibition inhibition (pM) inhibition 09823 5592 57 n.d. n.d. 09824 62 93 46 n.d. n.d. 09825 156 91 69 294 79 09974814 85 n.d. n.d. n.d. 10701 n.d. n.d. 59 n.d. n.d. 10702 n.d. n.d. 55n.d. n.d. 10703 70 89 62 449 79

In summary MOR09823, MOR09824, MOR09825, MOR09974, MOR10701, MOR10702MOR10703, MORI2609 and MORI2610 are each capable of inhibiting cellularHER3 activity in both a ligand-dependent and ligand-independent manner.

(ix) Inhibition of Proliferation

Since MOR09823, MOR09824, MOR09825, MOR09974, MOR10701, MOR10702 andMOR10703 all inhibited HER3 activity and downstream signaling they weretested for their ability to block ligand dependent and independent invitro cell growth (Example data is shown in FIG. 8 and summarized inTable 17). The anti-HER3 antibodies tested were all effective inhibitorsof cell proliferation.

TABLE 17 Inhibition of proliferation following treatment with 10 μg/mlanti-HER3 IgG in SK-Br-3, BT-474 and MCF7 cells. % Inhibition MOR#SK-Br-3 BT-474 MCF7 09823 39 39.8 82 09824 33 36.8 82 09825 41 37.2 6309974 35 n.d. 20 10701 n.d. 43.6 n.d. 10702 n.d. 43.8 n.d. 10703 35 41.681

(x) Ligand Blocking Assessment

The ability of the described anti-HER3 antibodies to block ligandbinding was assessed by examining the binding of neuregulin to MCF7cells previously treated with either MOR09823 or MOR09825. The presenceof either MOR09823 or MOR09825 had no significant effect upon theability of neuregulin to bind MCF7 cells whilst the positive controlused in the experiment (Mab3481) was capable of profoundly interferingwith neuregulin binding (see FIG. 9). These results are consistent withthe crystal structure since MOR09823 interacts with domains 2 and 4whilst the major contact points for HER3's interaction with neuregulinare hypothesized to be primarily clustered within domains 1 and 3. Giventhat neuregulin is capable of binding the inactive conformation of HER3(Kani et al., (2005) Biochemistry 44: 15842-15857) it is probable thatMOR09823 and MOR09825 function by preventing the HER3 domainrearrangements necessary for signaling or by interfering with receptordimerization.

(xi) Ligand Blocking Assessment (Biochemical)

To explore whether MOR09823 and neuregulin can bind HER3 concurrently abiochemical assay was established using Biacore™ technology. Interactionanalyses were performed by capturing biotinylated neuregulin on thesurface of a Biacore™ sensor chip CAP (GE Healthcare) utilizing a BiotinCAPture kit (GE Healthcare). HER3 complexes were generated by incubatinghuman HER3-Fc with increasing concentrations of either MOR09823, 105.5(Thermo Scientific) or human IgG. Preformed HER3/antibody complexes wereinjected over reference and active surfaces and the interaction of HER3with neuregulin observed.

Control IgG had no effect upon HER3/neuregulin complex formation whilst105.5 was observed to significantly inhibit the ability of HER3 to bindneuregulin confirming its description as a ligand blocking antibody(FIG. 10). In contrast HER3/MOR09823 complexes were capable of bindingneuregulin demonstrating that MOR09823 does not prevent ligand binding.Interestingly, a dose-dependent increase in RU values was uniquelyobserved when MOR09823/HER3 complexes were injected. This data indicatesthat a trimeric complex containing neuregulin, HER3 and MOR09823 isgenerated on the chip surface. The ability of this trimeric complex toform is predicted by the HER3/MOR09823 crystal structure since MOR09823binding does not occlude the ligand binding site of HER3 suggesting thatbinding of neuregulin and MOR09823 are not mutually exclusive.

In another embodiment, the antibody or fragment thereof binds to bothdomain 2 and domain 4 of HER3 and without blocking the concurrentbinding of a HER3 ligand such as neuregulin. While not required toprovide a theory, it is feasible that the antibody or fragment thereofbinding to both domain 2 and domain 4 of HER3, holds HER3 in an inactiveconformation without blocking the ligand binding site on HER3. Thus aHER3 ligand (e.g., neuregulin) is able to bind to HER3 at the same timeas the antibody.

The antibodies of the invention or fragments thereof inhibit both liganddependent and independent activation of HER3 without preventing ligandbinding. This is considered advantageous for the following reasons:

(i) The therapeutic antibody would have clinical utility in a broadspectrum of tumors than an antibody which targeted a single mechanism ofHER3 activation (i.e. ligand dependent or ligand independent) sincedistinct tumor types are driven by each mechanism.

(ii) The therapeutic antibody would be efficacious in tumor types whereboth mechanisms of HER3 activation are simultaneously involved. Anantibody targeting a single mechanism of HER3 activation (i.e. liganddependent or ligand independent) would display little or no efficacy inthese tumor types

(iii) The efficacy of an antibody which inhibits ligand dependentactivation of HER3 without preventing ligand binding would be lesslikely to be adversely affected by increasing concentrations of ligand.This would translate to either increased efficacy in a tumor type drivenby very high concentrations of HER3 ligand or a reduced drug resistanceliability where resistance is mediated by up-regulation of HER3 ligands.

(iv) An antibody which inhibits HER3 activation by stabilizing theinactive form would be less prone to drug resistance driven byalternative mechanisms of HER3 activation. Consequently, the antibodiesof the invention may be used to treat conditions where existingtherapeutic antibodies are clinically ineffective.

(xii) In Vivo Inhibition of HER3 Activity and Effect Upon Tumor Growth

To determine the in vivo activity of the described anti-HER3 antibodies,MOR09823 was tested in both BxPC-3 and BT-474 tumor models. MOR09823 wasdemonstrated to inhibit HER3 activity as evidenced by a significantreduction in tumor pHER3 levels (FIG. 11). Signaling downstream of HER3was similarly inhibited as demonstrated by reduced pAkt levels in bothBxPC-3 and BT-474 (FIG. 11). In a HER2 driven BT-474 efficacy study,repeated MORI0701 treatment yielded a 74% inhibition of tumor growth(see FIG. 12A) whilst MOR10703 yielded 83% inhibition. In the BxPC3tumor growth model, both MOR10701 and MORI0703 very effectivelyinhibited ligand driven tumor growth (see FIG. 13).

(xiii) In Vitro Drug Combinations and Impact Upon Cell Growth.

Since tumor cell growth is frequently driven by multiple signalingpathways we assessed whether combinations of MOR09823 or MOR10703 withvarious targeted agents would be of benefit in blocking cellproliferation. The targeted agents chosen primarily inhibited HER2(trastuzumab, lapatinib) EGFR (cetuximab, erlotinib), PI3K/mTOR(BEZ235), PI3K (BKM120), PIK3CA (BYL719) and mTOR (RAD001) since thesetargets are commonly activated in human tumors. Isobologram analysis(see FIG. 14) indicated that MOR09823 and MORI0703 displayed synergisticdrug combinations with trastuzumab, lapatinib, erlotinib, cetuximab,BEZ235, BKM120, BYL719 and RAD001. This data suggests that inhibition ofHER3 signaling is of particular benefit to inhibitors that targetreceptor tyrosine kinases or the PI3K signaling pathway.

(xiv) In Vivo MORI0703 Drug Combinations

Since HER3 inhibition combined with receptor tyrosine kinase targetedagents in vitro we assessed the impact of either MORI0701 or MORI0703 incombination with trastuzumab and erlotinib in vivo. In BT-474 xenografts(see FIG. 15A), combination of either MORI0701 or MORI0703 (20 mg/kg)with a sub-optimal dose of trastuzumab (1 mg/kg) was sufficient toinduce tumor regressions (% T/C=−50 and −37 respectively). In L3.3pancreatic xenografts, combination of MORI0703 (20 mg/kg) with dailyerlotinib (50 mg/kg) resulted in tumor stasis (% T/C=3, see FIG. 15B).In both models, the combination of two drugs was significantly moreefficacious than either drug alone thus supporting our earlier in vitrofinding of the benefit of combining HER3-targeted antibodies withErbB-targeted agents.

In summary, the unique ability of this family of antibodies to stabilizethe inactive conformation of HER3 results in significant in vivoefficacy in models where HER3 is activated in either a ligand dependentor independent manner. Furthermore, HER3 inhibition by this family ofantibodies appears beneficial in combination with a wide variety oftargeted therapies.

Example 22: HER3 Antibodies for Benign Prostatic Hyperplasia (BPH),Gynecomastia, and Endometriosis

Experimental Procedure

Sexually mature IGS Wistar Hannover rats of approximately 9 weeks of agewere dosed via intravenous injection on a twice weekly schedule with 30,75 and 200 mg/kg MOR0703. Following completion of the 13 week dosingperiod, 10 rats from each dose group were sacrificed and major organscollected for further analysis. In addition, 6 rats from the 200 mg/kgdose group were allowed to recover from MORI0703 for 10 weeks prior tosacrifice in order to determine the reversibility of any observedchanges. Post animal sacrifice, organ weights were recorded prior tofixation in 10% neutral-buffered formalin. Tissue sections were preparedand assessed by microscopic examination.

Results

In male rats, decreased prostate weight was observed at all doses, whichcorrelated with reduced secretion in the prostate and seminal vesicles(Table 18). These effects were reversible after 10 weeks of recovery.Differences in absolute mean vs. control were as follows: 30 mg/kg:−31%, 75 mg/kg: −40%, and 200 mg/kg: −35%. Mammary gland atrophy, mostlymoderate or marked, was present in all dosed males and was notreversible after 10 weeks recovery. This change was characterized by theabsence of the normally abundant acinar and lobular development seen inthe male mammary gland. By contrast to the controls, sparse ductularelements were present in the mammary glands of all treated males.

TABLE 18 MOR10703-related differences in organ weights in male rats SexMale Dose (mg/kg)  0  30  75 200 Number examined  10  10  10  10 Bodyweight mean (g) 412 390 392 397 (% diff) (—)  −5  −4  −4 Brain weightmean (g)  2.1  2.1  2.1  2.1 (% diff) (—)  −3  −3  −1 Prostate Absolutemean (g)  0.9  0.6 ^(c)  0.6 ^(c)  0.6 ^(c) (% diff) (—) −31 −40 −35Rel.^(b) to body weight (%)  0.22  0.16 ^(c)  0.14 ^(c)  0.15 ^(c) (%diff) (—) −28 −38 −33 Rel. to brain weight  43  31  27 ^(c)  28 ^(c) (%diff) (—) −29 −38 −35 ^(a)Expressed as percent difference of group means(% diff). ^(b)Relative. ^(c) Based upon statistical analysis of groupmeans, values are significantly different from control group.

In females, decreased uterus weight was observed at all doses, which wasreversible after 10 weeks of recovery (Table 19). Absolute mean vs.control differences were 30 mg/kg: −24%, 75 mg/kg: −27%, and 200 mg/kg:−19%. Endometrial atrophy, observed as a profound decrease in glandularepithelium in the uterus at all doses ≥30 mg/kg/day correlated with thedecrease in uterus weights observed at the end of treatment. This wasslightly reversible after weeks of recovery in that an increased amountof glandular epithelium was present although not as much as in controlanimals. Other reproductive organs: ovaries, oviducts, cervix and vaginaappeared to be within normal limits, considering the different stages ofthe cycle that can be observed.

TABLE 19 MOR10703-related differences in organ weights in female ratsSex Female Dose (mg/kg)  0  30  75 200 Number examined  10  10  10  10Body weight mean (g) 237 246 239 241 (% diff) (—)  −4  −1  −2 Brainweight mean (g)  1.9  2.0  1.9  2.0 (% diff) (—)  −1  −1  −2 UterusAbsolute mean (g)  0.48  0.36 ^(c)  0.35 ^(c)  0.39 (% diff) (—) −24 −27−19 Rel.^(b) to body weight (%)  0.20  0.15 ^(c)  0.15 ^(c)  0.16 (%diff) (—) −26 −27 −21 Rel. to brain weight  25  18 ^(c)  18 ^(c)  20^(c) (% diff) (—) −29 −38 −35 ^(a)Expressed as percent difference ofgroup means (% diff). ^(b)Relative. ^(c) Based upon statistical analysisof group means, values are significantly different from control group.Refer to data tables for actual significance levels and tests used.

Discussion

Benign prostatic hyperplasia (BPH) is a common disease in aging malesthat is characterized by a non-neoplastic enlargement of the prostateleading to pressure on the urethra and resulting in urination andbladder problems {Mahapokail W, van Sluijs F J & Schalken J A. 2000Prostate Cancer and Prostatic Diseases 3, 28-33}. Anatomic ormicroscopic evidence of BPH is present at autopsy in approximately 55%of men aged 60-70 years. Transurethral resection of the prostate hasbeen the treatment of choice for many years. Consequently, BPH is one ofthe most common reasons for surgical intervention among elderly men.Less invasive methods of treatment include:

(i) Alpha 1-blockers (doxazosin, prazosin, tamsulosin, terazosin, andalfuzosin) are a class of medications also used to treat high bloodpressure. These medications relax the muscles of the bladder neck andprostate thus allowing easier urination.

(ii) Finasteride and dutasteride lower androgen levels thus reducing thesize of the prostate gland, increasing urine flow rate, and decreasingsymptoms of BPH. It may take 3 to 6 months before improvement insymptoms is observed. Potential side effects related to the use offinasteride and dutasteride include decreased sex drive and impotence

The results in the Experiments section demonstrate for the first timethat BPH is a neuregulin-dependent indication. The finding that MOR10703significantly reduced prostate size in sexually mature rats withoutaffecting hormone levels suggests that MOR10703 could be useful for thetreatment of BPH.

To further test MOR10703 and other HER3 antibodies as therapeutics forBPH, primary human BPH surgical specimens can be transplanted intoathymic mice or rats and effect of the Her3 antibodies studied usingmodels (Otto U et al. Urol Int 1992; 48: 167-170). Alternatively,aspects of BPH can be induced in castrated dogs via the long-termadministration of 5α-Androstane-3a, 170-diol plus estradiol and the HER3antibodies tested in these canine models (Walsh P C, Wilson J D. J ClinInvest 1976; 57: 1093-1097).

The physical manifestation of gynecomastia is breast enlargement inmales, it normally occurs in both breasts but can sometimes be in oneonly, known as asymmetrical or unilateral gynecosmastia. Gynecomastia iscommonly caused by:

(i) Elevated estrogen levels resulting in the ratio of testosterone tooestrogen becoming unbalanced.

(ii) Androgen antagonists or anti-androgens used in the treatment ofprostate cancer or BPH. These drugs suppress testosterone but bysuppressing testosterone, oestrogen begins to rise.

Although a number of experimental drugs are currently being evaluatedthere are currently no approved treatments for gynecomastia.Consequently gynecomastia is treated via surgical removal of the breasttissue. The observation that MOR10703 induced irreversible atrophy ofthe male mammary gland indicates that it may be of benefit in thetreatment of gynecomastia.

To further test MOR10703 and other HER3 antibodies for treating humangynecomastia, transgenic mouse models of gynecomastia can be used. Thesemodels have been developed by expressing human aromatase in the mousemammary gland and recapitulate many aspects of human gynecomastia (Li etal., Endocrinology 2002; 143:4074-4083; Tekmal et al., Cancer Res 1996;56:3180-318).

MOR10703 and other HER3 antibodies can also be examined for treatingendometriosis, a gynecological condition in which cells from the liningof the uterus (endometrium) appear and flourish outside the uterinecavity. The main, but not universal, symptom of endometriosis is pelvicpain in various manifestations. Although the underlying causes ofendometriosis are not well characterized is thought to be dependent onthe presence of estrogen. Since MOR10703 induced endometrial atrophy infemale mice resulting in a decrease in uterus weight, it may be used totreat endometriosis. To further study the effects of MOR10703 and otherHER3 antibodies on endometriosis, human endometrium in the proliferativephase can be implanted into the peritoneal cavity of normal cycling andovariectomized athymic mice or cycling non-obese diabetic (NOD)-severecombined immuno-deficiency (SCID) mice, and these SCID mice used asmodels (Grummer et al., 2001. Human Reproduction; 16; 1736-1743).

Example 23: In Vitro Studies to Assess HER3 Antibodies for EsophagealCancer

In Vitro Esophageal Drug Combination Studies

To assess the ability of HER3-targeted antibodies to combine withtargeted therapies MOR10703 was combined with cetuximab or BYL719 incell viability assays. Approximately 1000-1200 KYSE140 and KYSE180 cellswere seeded into 384-well plates in the appropriate culture mediasupplemented with 2% FBS and allowed to adhere overnight at 37° C. Theappropriate drug combinations (typical final drug concentrations forBYL719 ranged from 2.8 μM to 3.8 nM; for cetuximab ranged from 93 nM to0.13 nM; for MOR10703 100 nM to 4.1 nM) were subsequently added to thewells such that each plate contained a dose response curve of each drugin a two-dimensional matrix. Treated cells were subsequently incubatedfor 96-120 hours. At the end of the drug treatment, CellTiter-Glo regentwere added to each well to lyse the cells, and luminescence signals wererecorded using an Envision plate reader. The extent of growth inhibitionobtained with each combination was calculated and combination activityhighlighted using the Loewe additivity model.

Esophageal In Vitro Drug Combinations and Impact Upon Cell Growth

Since tumor cell growth is frequently driven by multiple signalingpathways, combinations of MOR10703 with cetuximab or BYL719 wereassessed to determine whether they would be of benefit in blockingproliferation of esophageal cancer cell lines. The targeted agentschosen primarily inhibited EGFR (cetuximab) and PIK3CA (BYL719) sincethese targets are commonly activated in human tumors. Isobologramanalysis (see FIG. 16) indicated that MOR10703 displayed synergisticdrug combinations with cetuximab and BYL719. This data shows thatinhibition of HER3 signaling is of particular benefit to inhibitors thattarget receptor tyrosine kinases or the PI3K signaling pathway.

Example 24: In Vivo Studies to Assess HER3 Antibodies for EsophagealCancer

To assess the in vivo ability of HER3-targeted antibodies to combinewith targeted therapies MOR10703 was combined with cetuximab or BYL719and tested in two in vivo xenograft models.

(i) In Vivo KYSE140 Xenografts

KYSE140 cells were cultured in RPMI 1640 medium containing 10%heat-inactivated fetal bovine serum without antibiotics until the timeof implantation. KYSE140 cells were harvested in exponential growth. Tenmillion cells were mixed with PBS/Matrigel (50:50) were subcutaneouslyimplanted into the upper right flank of SCID-Beige mice. On Day 28,tumors were measured and animals with a tumor volume of approximately200 mm³ were enrolled in the efficacy study. In general, a total of 10animals per group were enrolled in efficacy studies. For single-agentand combination studies, animals were dosed intravenously via lateraltail vein injection with MOR10703 or cetuximab. BYL719 was formulated in0.5% methylcellulose and dosed via oral gavage.

(ii) In Vivo KYSE180 Xenografts

KYSE180 cells were cultured in RPMI 1640 medium containing 10%heat-inactivated fetal bovine serum without antibiotics until the timeof implantation. KYSE180 cells were harvested in exponential growth.Five million cells were subcutaneously implanted into the upper rightflank of nude mice. On Day 18, tumors were measured and animals with atumor volume of approximately 200 mm³ were enrolled in the efficacystudy. In general, a total of 10 animals per group were enrolled inefficacy studies. For single-agent and combination studies, animals weredosed intravenously via lateral tail vein injection with MOR10703 orcetuximab. BYL719 was formulated in 0.5% methylcellulose and dosed viaoral gavage.

(iii) In Vivo Primary Esophageal Xenografts

Human esophageal primary tumors were passaged in mice. When tumor sizereached approximately 150 mm³ in size, animals were enrolled in theefficacy study. For single-agent and combination studies, animals weredosed intravenously via lateral tail vein injection with MOR10703 orcetuximab. BYL719 was formulated in 0.5% methylcellulose and dosed viaoral gavage.

In Vivo Inhibition of HER3 and Effect Upon Esophageal Tumor Growth

To determine the in vivo activity of the described anti-HER3 antibodies,MOR10703 was tested in both KYSE140 and KYSE180 esophageal tumor models,as well as two primary esophageal tumor models, CHES007 and CHES015. Inboth KYSE140 and KYSE180 in vivo models, treatment with single-agentMOR10703 was demonstrated to effectively inhibit tumor growth (FIG. 17).In KYSE180, the combination of MOR10703 and BYL719 was sufficient toinduce significant tumor regressions. These findings were extended tothe primary esophageal tumor models, where a combination of MORI0703with either cetuximab or BYL719 also induced potent tumor regressions(FIG. 18). Together, these data further support our earlier in vitrofinding of the benefit of combining HER3-targeted antibodies with witherEGFR or PI3K-targeted agents.

Example 25: In Vivo Studies to Assess HER3 Combinations with BYL719 inGastric Cancer

To assess the in vivo ability of HER3-targeted antibodies to combinewith targeted therapies in gastric cancer, MORI0703 was combined withBYL719 and tested in an in vivo xenograft model.

(i). In Vivo NCI-N87 Xenograft

NCI-N87 cells were grown in DMEM culture medium containing 4.5 g/lglucose supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 1mM sodium pyruvate until the point of implantation. NCI-N87 tumors wereestablished by injecting 8×106 to 1×107 cells (in HBSS containing 50%v/v Matrigel) subcutaneously. On Day 10, tumors were measured andanimals with a tumor volume of approximately 250 mm³ were enrolled inthe efficacy study. For single-agent and combination arms, animals weredosed intravenously via lateral tail vein injection with MOR10703.BYL719 was formulated in 0.5% methylcellulose and dosed via oral gavage.

Effect of Combination Treatment on N87 Gastric Tumor Growth

As seen was seen for esophageal tumors, the combination of MOR10703 andBYL719 was sufficient to induce significant, prolonged tumor regressionsin the N87 gastric tumor model thus further supporting the benefit ofcombining HER3-targeted antibodies with PI3K-targeted agents (see FIG.19).

Example 26: In Vivo Studies to Assess HER3 Combinations with Cetuximabin Squamous Cell Cancer of the Head and Neck (SCCHN)

To assess the in vivo ability of HER3-targeted antibodies to combinewith targeted therapies in SCCHN, MOR10703 was combined with cetuximaband tested in an in vivo xenograft model.

(i). In Vivo A253 Xenograft

A253 cells were cultured in DMEM containing 10% heat-inactivated fetalbovine serum without antibiotics until the time of implantation. A253cells were harvested in exponential growth. Five million cells in 200 μlPBS were subcutaneously implanted into the upper right flank of nudemice. On Day 25, tumors were measured and animals with a tumor volume ofapproximately 200 mm³ were enrolled in the efficacy study. In general, atotal of 9 animals per group were enrolled in efficacy studies. Forsingle-agent and combination studies, animals were dosed intravenouslyvia lateral tail vein injection with MOR10703 or cetuximab.

Effect of Combination Treatment on A253 SCCHN Tumor Growth

In the A253 SCCHN model, treatment with either MOR10703 or cetuximab asa single agent resulted in tumor stasis. Combination of MOR10703 withcetuximab was significantly more active and resulted in tumor regression(see FIG. 20).

Collectively, these results show MORI0703 as a single agent can inhibittumor growth. The results also show there is a synergistic effect ontumor regression when MORI0703 is combined with inhibitors that targetother receptor tyrosine kinases or the PI3K signaling pathway.

INCORPORATION BY REFERENCE

All references cited herein, including patents, patent applications,papers, text books, and the like, and the references cited therein, tothe extent that they are not already, are hereby incorporated herein byreference in their entirety.

EQUIVALENTS

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The foregoingdescription and examples detail certain preferred embodiments of theinvention and describe the best mode contemplated by the inventors. Itwill be appreciated, however, that no matter how detailed the foregoingmay appear in text, the invention may be practiced in many ways and theinvention should be construed in accordance with the appended claims andany equivalents thereof.

We claim:
 1. A method of treating a disorder characterized by increasedlevels of HER3 expression in an esophageal tract comprising: selectingpatient suffering from increased levels of HER3 expression in anesophageal tract; and administering an antibody or fragment thereof thatspecifically binds to a HER3 receptor, such that the antibody orfragment thereof binds to a conformational epitope comprising amino acidresidues within domain 2 and domain 4 of the HER3 receptor and blocksboth ligand-dependent and ligand-independent signal transduction,thereby treating the disorder.
 2. The method of claim 1, wherein thedisorder is selected from the group consisting of esophageal cancer andBarretts esophageal cancer.
 3. The method of claim 1, wherein theantibody or fragment thereof is administered by a route selected fromthe group consisting of oral, subcutaneous, intraperitoneal,intramuscular, intracerebroventricular, intraparenchymal, intrathecal,intracranial, buccal, mucosal, nasal, and rectal administration.
 4. Themethod of claim 1, wherein the antibody or fragment is formulated into apharmaceutical composition comprising a physiologically acceptablecarrier, excipient, or diluent.
 5. The method of claim 4, furthercomprising an additional therapeutic agent.
 6. The method of claim 5,wherein the additional therapeutic agent is selected from the groupconsisting of an HER1 inhibitor, a HER2 inhibitor, a HER3 inhibitor, aHER4 inhibitor, an mTOR inhibitor and a PI3 Kinase inhibitor.
 7. Themethod of claim 6, wherein the additional therapeutic agent is a HER1inhibitor selected from the group consisting of Matuzumab (EMD72000),Erbitux®/Cetuximab, Vectibix®/Panitumumab, mAb 806, Nimotuzumab,Iressa®/Gefitinib, CI-1033 (PD183805), Lapatinib (GW-572016),Tykerb®/Lapatinib Ditosylate, Tarceva®/Erlotinib HCL (OSI-774), PKI-166,and Tovok®; a HER2 inhibitor selected from the group consisting ofPertuzumab, Trastuzumab, MM-111, neratinib, lapatinib or lapatinibditosylate/Tykerb®; a HER3 inhibitor selected from the group consistingof, MM-121, MM-111, IB4C3, 2DID12 (U3 Pharma AG), AMG888 (Amgen), AV-203(Aveo), MEHD7945 Å (Genentech), MOR10703 (Novartis) and small moleculesthat inhibit HER3; and a HER4 inhibitor.
 8. The method of claim 6,wherein the additional therapeutic agent is an mTOR inhibitor selectedfrom the group consisting of Temsirolimus/Torisel®,ridaforolimus/Deforolimus, AP23573, MK8669, everolimus/Affinitor®. 9.The method of claim 6, wherein the additional therapeutic agent is a PI3Kinase inhibitor selected from the group consisting of GDC 0941, BEZ235,BMK120 and BYL719.
 10. A method of treating gastric cancer comprising:selecting a patient suffering from gastric cancer; and administering anantibody or fragment thereof that specifically binds to a HER3 receptor,such that the antibody or fragment thereof binds to a conformationalepitope comprising amino acid residues within domain 2 and domain 4 ofthe HER3 receptor and blocks both ligand-dependent andligand-independent signal transduction, thereby treating gastric cancer.11. The method of claim 10, wherein the antibody or fragment thereof isadministered by a route selected from the group consisting of oral,subcutaneous, intraperitoneal, intramuscular, intracerebroventricular,intraparenchymal, intrathecal, intracranial, buccal, mucosal, nasal, andrectal administration.
 12. The method of claim 10, wherein the antibodyor fragment is formulated into a pharmaceutical composition comprising aphysiologically acceptable carrier, excipient, or diluent.
 13. Themethod of claim 12, further comprising an additional therapeutic agent.14. The method of claim 13, wherein the additional therapeutic agent isselected from the group consisting of an HER1 inhibitor, a HER2inhibitor, a HER3 inhibitor, a HER4 inhibitor, an mTOR inhibitor and aPI3 Kinase inhibitor.
 15. The method of claim 14, wherein the additionaltherapeutic agent is a HER1 inhibitor selected from the group consistingof Matuzumab (EMD72000), Erbitux®/Cetuximab, Vectibix®/Panitumumab, mAb806, Nimotuzumab, Iressa®/Gefitinib, CI-1033 (PD183805), Lapatinib(GW-572016), Tykerb®/Lapatinib Ditosylate, Tarceva®/Erlotinib HCL(OSI-774), PKI-166, and Tovok®; a HER2 inhibitor selected from the groupconsisting of Pertuzumab, Trastuzumab, MM-111, neratinib, lapatinib orlapatinib ditosylate/Tykerb®; a HER3 inhibitor selected from the groupconsisting of, MM-121, MM-111, IB4C3, 2DID12 (U3 Pharma AG), AMG888(Amgen), AV-203 (Aveo), MEHD7945 Å (Genentech), MORI0703 (Novartis) andsmall molecules that inhibit HER3; and a HER4 inhibitor.
 16. The methodof claim 14, wherein the additional therapeutic agent is an mTORinhibitor selected from the group consisting of Temsirolimus/Torisel®,ridaforolimus/Deforolimus, AP23573, MK8669, everolimus/Affinitor®. 17.The method of claim 14, wherein the additional therapeutic agent is aPI3 Kinase inhibitor selected from the group consisting of GDC 0941,BEZ235, BMK120 and BYL719.
 18. A method of treating head and neck cancercomprising: selecting a patient suffering from cancer the head and neck;and administering an antibody or fragment thereof that specificallybinds to a HER3 receptor, such that the antibody or fragment thereofbinds to a conformational epitope comprising amino acid residues withindomain 2 and domain 4 of the HER3 receptor and blocks bothligand-dependent and ligand-independent signal transduction, therebytreating head and neck cancer.
 19. The method of claim 18, wherein theantibody or fragment thereof is administered by a route selected fromthe group consisting of oral, subcutaneous, intraperitoneal,intramuscular, intracerebroventricular, intraparenchymal, intrathecal,intracranial, buccal, mucosal, nasal, and rectal administration.
 20. Themethod of claim 18, wherein the antibody or fragment is formulated intoa pharmaceutical composition comprising a physiologically acceptablecarrier, excipient, or diluent.
 21. The method of claim 20, furthercomprising an additional therapeutic agent.
 22. The method of claim 21,wherein the additional therapeutic agent is selected from the groupconsisting of an HER1 inhibitor, a HER2 inhibitor, a HER3 inhibitor, aHER4 inhibitor, an mTOR inhibitor and a PI3 Kinase inhibitor.
 23. Themethod of claim 22, wherein the additional therapeutic agent is a HER1inhibitor selected from the group consisting of Matuzumab (EMD72000),Erbitux®/Cetuximab, Vectibix®/Panitumumab, mAb 806, Nimotuzumab,Iressa®/Gefitinib, CI-1033 (PD183805), Lapatinib (GW-572016),Tykerb®/Lapatinib Ditosylate, Tarceva®/Erlotinib HCL (OSI-774), PKI-166,and Tovok®; a HER2 inhibitor selected from the group consisting ofPertuzumab, Trastuzumab, MM-111, neratinib, lapatinib or lapatinibditosylate/Tykerb®; a HER3 inhibitor selected from the group consistingof, MM-121, MM-111, IB4C3, 2DID12 (U3 Pharma AG), AMG888 (Amgen), AV-203(Aveo), MEHD7945 Å (Genentech), MORI0703 (Novartis) and small moleculesthat inhibit HER3; and a HER4 inhibitor.
 24. The method of claim 22,wherein the additional therapeutic agent is an mTOR inhibitor selectedfrom the group consisting of Temsirolimus/Torisel®,ridaforolimus/Deforolimus, AP23573, MK8669, everolimus/Affinitor®. 25.The method of claim 22, wherein the additional therapeutic agent is aPI3 Kinase inhibitor selected from the group consisting of GDC 0941,BEZ235, BMK120 and BYL719.
 26. A method of treating gynacomastica,comprising: selecting a patient suffering from gynacomastica; andadministering an antibody or fragment thereof that specifically binds toa HER3 receptor, such that the antibody or fragment thereof binds to aconformational epitope comprising amino acid residues within domain 2and domain 4 of the HER3 receptor and blocks both ligand-dependent andligand-independent signal transduction, thereby treating gynacomastica.27. The method of claim 26, wherein the antibody or fragment thereof isadministered by a route selected from the group consisting of oral,subcutaneous, intraperitoneal, intramuscular, intracerebroventricular,intraparenchymal, intrathecal, intracranial, buccal, mucosal, nasal, andrectal administration.
 28. The method of claim 26, wherein the antibodyor fragment is formulated into a pharmaceutical composition comprising aphysiologically acceptable carrier, excipient, or diluent.
 29. Themethod of claim 26, further comprising an additional therapeutic agent.30. The method of claim 29, wherein the additional therapeutic agent isselected from the group consisting of an HER1 inhibitor, a HER2inhibitor, a HER3 inhibitor, a HER4 inhibitor, an mTOR inhibitor and aPI3 Kinase inhibitor.
 31. The method of claim 30, wherein the additionaltherapeutic agent is a HER1 inhibitor selected from the group consistingof Matuzumab (EMD72000), Erbitux®/Cetuximab, Vectibix®/Panitumumab, mAb806, Nimotuzumab, Iressa®/Gefitinib, CI-1033 (PD183805), Lapatinib(GW-572016), Tykerb®/Lapatinib Ditosylate, Tarceva®/Erlotinib HCL(OSI-774), PKI-166, and Tovok®; a HER2 inhibitor selected from the groupconsisting of Pertuzumab, Trastuzumab, MM-111, neratinib, lapatinib orlapatinib ditosylate/Tykerb®; a HER3 inhibitor selected from the groupconsisting of, MM-121, MM-111, IB4C3, 2DID12 (U3 Pharma AG), AMG888(Amgen), AV-203 (Aveo), MEHD7945 Å (Genentech), MORI0703 (Novartis) andsmall molecules that inhibit HER3; and a HER4 inhibitor.
 31. The methodof claim 30, wherein the additional therapeutic agent is an mTORinhibitor selected from the group consisting of Temsirolimus/Torisel®,ridaforolimus/Deforolimus, AP23573, MK8669, everolimus/Affinitor®. 32.The method of claim 30, wherein the additional therapeutic agent is aPI3 Kinase inhibitor selected from the group consisting of GDC 0941,BEZ235, BMK120 and BYL719.
 33. A method of treating endometriosiscomprising: selecting a patient suffering from endometriosis; andadministering an antibody or fragment thereof that specifically binds toa HER3 receptor, such that the antibody or fragment thereof binds to aconformational epitope comprising amino acid residues within domain 2and domain 4 of the HER3 receptor and blocks both ligand-dependent andligand-independent signal transduction, thereby treating endometriosis.34. The method of claim 33, wherein the antibody or fragment thereof isadministered by a route selected from the group consisting of oral,subcutaneous, intraperitoneal, intramuscular, intracerebroventricular,intraparenchymal, intrathecal, intracranial, buccal, mucosal, nasal, andrectal administration.
 35. The method of claim 34, wherein the antibodyor fragment is formulated into a pharmaceutical composition comprising aphysiologically acceptable carrier, excipient, or diluent.
 36. Themethod of claim 35, further comprising an additional therapeutic agent.37. The method of claim 36, wherein the additional therapeutic agent isselected from the group consisting of an HER1 inhibitor, a HER2inhibitor, a HER3 inhibitor, a HER4 inhibitor, an mTOR inhibitor and aPI3 Kinase inhibitor.
 38. The method of claim 37, wherein the additionaltherapeutic agent is a HER1 inhibitor selected from the group consistingof Matuzumab (EMD72000), Erbitux®/Cetuximab, Vectibix®/Panitumumab, mAb806, Nimotuzumab, Iressa®/Gefitinib, CI-1033 (PD183805), Lapatinib(GW-572016), Tykerb®/Lapatinib Ditosylate, Tarceva®/Erlotinib HCL(OSI-774), PKI-166, and Tovok®; a HER2 inhibitor selected from the groupconsisting of Pertuzumab, Trastuzumab, MM-111, neratinib, lapatinib orlapatinib ditosylate/Tykerb®; a HER3 inhibitor selected from the groupconsisting of, MM-121, MM-111, IB4C3, 2DID12 (U3 Pharma AG), AMG888(Amgen), AV-203 (Aveo), MEHD7945 Å (Genentech), MORI0703 (Novartis) andsmall molecules that inhibit HER3; and a HER4 inhibitor.
 39. The methodof claim 37, wherein the additional therapeutic agent is an mTORinhibitor selected from the group consisting of Temsirolimus/Torisel®,ridaforolimus/Deforolimus, AP23573, MK8669, everolimus/Affinitor®. 40.The method of claim 39, wherein the additional therapeutic agent is aPI3 Kinase inhibitor selected from the group consisting of GDC 0941,BEZ235, BMK120 and BYL719.